COMPOSITION AND ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS OF SALVIA FRUTICOSA AND SALVIA RINGENS (LAMIACEAE) SASTAV I ANTIMIKROBNA AKTIVNOST ETARSKIH ULJA SALVIA FRUTICOSA I SALVIA RINGENS (LAMIACEAE)

Background/Aim. Plant essential oils (EOs) can have a significant antibacterial effect especially through additive or synergistic action as antibiotic adjuvants. We investigated the composition and activity of EOs of two species of genus Salvia from Greece with an aim to evaluate their antimicrobial activity as well as the activity of essential oil in combination with selected antibiotics. Methods. The aerial parts of wild-growing S. fruticosa and S. ringens were subjected to a steam distillation and the obtained EOs were analyzed by gas chromatography and gas chromatography/mass spectrometry. The broth-microdilution method was used in order to determine the minimum inhibitory concentrations (MICs) of essential oils on seven strains of bacteria and one yeast. The antimicrobial activity of the combination of essential oil and antibiotics was investigated by checkerboard method and estimated by calculating the fractional inhibitory concentration (FIC) of each component and the fractional inhibitory concentration index (FICI). Results. Dominant component of S. fruticosa essential oil was trans -thujone (54.2%), and for S. ringens essential oil it was α-pinene (28.1%). The MICs of essential oils of both species were in the range from 200 μg/ml to >500 μg/ml. The strongest antimicrobial effect was achieved against B. subtilis and C. albicans. According to FICI values essential oil of S. fruticosa had additive effect with ciprofloxacin against most of bacterial strains but not with amikacin. Conclusion . The essential oils of S. ringens and S. fruticosa showed modest antimicrobial activity, however essential oil of S. fruticosa showed a promising additive effect in combination with ciprofloxacin.


Introduction
Excessive and inappropriate use of antibiotics in the treatment of various infectious diseases has led to the resistance of many pathogens, which is a global problem in healthcare practice today, recorded also in Serbia 1 . In order to find new antimicrobial agents that would help to decrease the use of antibiotics, research into traditionally used medicinal plants and herbal products as well as related species of known medicinal plants has been intensified.
Earlier studies have shown that essential oils as mixtures of different plant secondary metabolites can have a significant antimicrobial effect, which is based on a different mechanism of action than antibiotics 2 . One possible approach to the use of essential oils in therapy is their use as an antibiotic adjuvant, with the aim of achieving a multitarget effect on pathogens through additive or synergistic action 3 .
Many studies on the anti-multidrug-resistant bacteria activity of aromatic members of Lamiaceae family are performed, primarily on essential oil of lavender, mint and thyme 2 .
Recently, it has been proved that the essential oil isolated from young leaves of Dalmatian sage (Salvia officinalis L.) also has an antimicrobial effect on certain human pathogens, and that the use of essential oil potentiated the inhibitory effect of antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) 4 .
Within genus Salvia, comprised of about 1000 worldwide distributed species, besides S. officinalis only several species with medicinal properties are aromatic plants with a significant amount of essential oil 5 . The essential oil of S. tomentosa, with β-pinene, αpinene and camphor as the main components, showed antimicrobial activity against a panel of microorganisms 6 . A study of composition and antimicrobial effects of essential oil and extracts of S. ringens from North Macedonia rich with 1,8-cineole, camphene, and borneol has shown that Gram-positive strains were more sensitive to essential oil 7 . The essential oil of S. amplexicaulis characterized by a high amount of sesquiterpenes, with germacrene D, viridiflorol, caryophyllene oxide and β-caryophyllene being the main components, showed inhibitory properties against Gram-positive bacteria and a yeast Candida albicans 8

. The
Greek sage, S. fructicosa, as the most widespread sage species in Mediterranean, has been used for its healing properties since the ancient times 5 . The oil of this species with high contents of 1,8-cineole, α-and β-thujone, and camphor, was tested for antimicrobial activity against eight common bacterial strains 9 , as well as against human pathogenic yeast 10 . The combined effect of tetracycline and essential oils of S officinalis, S. sclarea and S. fruticosa against clinical isolates of methicillin resistant Staphylococcus epidermidis showed synergistic or additive effects 11,12 . However, there are no consistent results of the antimicrobial activity of the combination of essential oil of S. fruticosa and antibiotics with different mechanism of action and different standard bacterial strains.
Knowing that the composition of essential oil of Salvia species differs throughout different developmental stages 13 and that it is highly influenced by the climatic conditions, we investigated the composition and activity of essential oil of two species of genus Salvia from Greece (S. fruticosa and S. ringens) collected during the late flowering stage with aim to evaluate their antimicrobial activity as well as activity of essential oil of S. fruticosa in combination with antibiotics (amikacin and ciprofloxacin).  For the isolation and analysis of the essential oil, air-dried plant material was subjected to a 2 hour steam distillation in a Clevenger-type apparatus according to Ph.Eur. 8.0 16 .

The plant material was sampled in Greece in
Qualitative analysis of the essential oils was performed using analytical gas chromatography (GC/FID) and gas chromatography/mass spectrometry (GC/MS). GC/FID and GC/MS analyses were carried out using an Agilent 6890N GC system equipped with The FIC of each compound was calculated by dividing the concentration of the compound in effective MIC of the combination with the MIC of the drug alone (e.g. FIC essential oil = MIC essential oil-antibiotic combination /MIC essential oil ). FICI values were calculated as the sum of the FIC essential oil and FIC antibiotic and interpreted as following: FICI ≤ 0.5 synergy; 0.5 < FICI ≤ 1 additivity; 1 < FICI ≤ 2 indifference (no effect) and FICI ≥ 2 antagonism 19,20 .
The minimum inhibitory concentrations of S. fruticosa and S. ringens essential oils were in the range from 200 μg/ml to >500 μg/ml ( Table 3). The antimicrobial activity of investigated oils was modest against most of the selected bacteria when compared to antibiotics. The greatest antibacterial activity was noted against B. subtilis, with MICs of 200 and 250 µg/ml of S. ringens and S. fruticosa oil, respectively. Also, C. albicans growth was inhibited by 300 µg/ml and 200 µg/ml of essential oils of the listed plant species.
Due to the small quantities of S. ringens aerial parts essential oil, further investigation of combined oil/antibiotics antimicrobial activity was performed with more abundant essential oil of S. fruticosa.
The antimicrobial activity of combination of essential oil and antibiotics is presented in Table 4. According to FICI values there is a significant difference in contribution of S. fruticosa essential oil to antimicrobial activity of antibiotics with different mechanism of action (amikacin and ciprofloxacin). With ciprofloxacin the additive effect (AD) was observed for all bacterial strains except for standard strain of Acinetobacter baumannii against which the combination was found to be indifferent (IN). The combination of essential oil and amikacin yielded no significant results, as it was indifferent against all strains except K. pneumoniae where the antagonistic effect was manifested.

Discussion
Previous investigations have shown a highly variable composition of essential oil of S. fruticosa, even when it comes to samples from similar habitats. In early study of composition and antimicrobial activity of S. fruticosa oil, the Greek samples had 1,8cineole (47.48%), thujone (11.93%), and camphor (9.04%) as the main components, while our sample had trans-thujone (54.2%) as a dominant compound and a low amount of 1,8cineole (0.2%). In the same study, investigation of antimicrobial activity of oil and its main compounds by disk diffusion assay showed relatively low levels of antimicrobial activity against the bacteria tested 9 .Taking into account the differences between antimicrobial assessment methods, the similar results were presented by Khoury et al. for S. fruticosa essential oil from Lebanon. The investigation was performed using broth-microdilution method and obtained MICs were greater than 500 μg/ml for S. aureus, E. coli and C.
albicans which is consistent with our findings 21 .
The overall composition of S. ringens essential oil is also variable, according to the results given by Alimpić et al. when compared with our results. While the main constituents of S. ringens oil from Greece in our study were α-pinene (28.1%), β-pinene (12.2%) and 1,8cineole (13%), the oil obtained from areal parts of the plant from North Macedonia was rich with 1.8-cineole (32.0%), camphene (17.1%) and borneol (11.9%) 7 . In the same study the moderate antibacterial activity of oil was shown against Gram-positive bacteria, especially S. aureus, and was attributed to high content of 1,8-cineole. Considering the lower content of this monoterpene in our sample, the results of antibacterial activity in our study are expected.
Given that the combined antibacterial activity of S. fruticosa oil with amikacin or ciprofloxacin was not tested before, we used these antibiotics in our study 3   RI -Linear retention indices determined in relation to a homologous series of n-alkanes (C 9 -C 24 ); ttrace.  Table 4 The activity of S. fruticosa essential oil in combination with antibiotics