ANTIBACTERIAL PROFILE OF PEUCEDANUM LONGIFOLIUM ESSENTIAL OIL

In the present study, the chemical composition and antibacterial activity of Peucedanum longifolium Waldst. & Kit. (Apiaceae) essential oil were examined, as well as the association between it and standard antibiotics. Gas chromatography and gas chromatography/mass spectrometry were used to analyze the chemical composition of the oil. The antibacterial activity of the oil was investigated by the broth microdilution method against thirteen bacterial strains. The interactions of the essential oil with three conventional antibiotics: tetracycline, streptomycin and chloramphenicol toward five selected bacterial strains were evaluated using the microdilution checkerboard assay. Monoterpene hydrocarbons (61.60%), with myrcene (15.88%) as the dominant constituent, were the most abundant compound class of the essential oil of P. longifolium from Serbia. The researched essential oil exhibited slight antibacterial activity against the tested bacterial strains in vitro. On the contrary, essential oil of P. longifolium posseses significant synergistic potential in combination with streptomycin and chloramphenicol (FIC indices in the range 0.21–0.87). Their combinations reduced the minimum effective dose of the antibiotic and, consequently, minimized its adverse side effects. In addition, investigated interactions are especially successful against Gram-negative bacteria, the pharmacological treatment of which is very difficult nowadays. These results indicate a method to enhance the efficacy of antibacterial drugs, especially against resistant bacterial strains. Acta Medica Medianae 2015; 54(1): 20-26.


Introduction
Bacterial resistance to antimicrobial drugs is a major obstacle to the treatment of infectious diseases (1). Thus, a search for new and more sustainable antibiotics is a necessity. Many studies have shown significant antibacterial activity of essential oils against a wide range of resistant microbial strains (2,3). To enhance the efficacy of antibacterial drugs and to reduce the required concentration, their combined use with essential oils is one of the promising strategies (4, 5).
The genus Peucedanum belongs to the Apiaceae family and consists of 120 economically important species that are used as foods and pharmaceuticals (6). Peucedanum longifolium Waldst. & Kit. is one of 14 species of this genus growing wild in Serbia (7). The search of the available literature revealed no data on the antibacterial activity of P. longifolium essential oil, especially in the combination essential oil-conventional antibiotic.

Aim
Given the importance of Peucedanum species, the aim of the present study was to examine the chemical composition and antibacterial activity of P. longifolium essential oil, as well as its combination with standard antibiotics: tetracycline, streptomycin and chloramphenicol.

Plant material and chemicals
The aerial parts of Peucedanum longifolium Waldst. & Kit. (Apiaceae) were collected in July 2011 from natural populations at the Rtanj mountain, Serbia. A voucher specimen, with the www.medfak.ni.ac.rs/amm accession number 16537 is deposited at the Herbarium of the Department of Botany, Faculty of Biology, University of Belgrade-Herbarium Code BEOU. All chemicals, reagents and standards were of analytical reagent grade and were purchased from the Sigma-Aldrich Chemical Company.
Oil isolation, gas chromatography, gas chromatography/mass spectrometry and identification of compounds.
Oil isolation, GC, GC-MS analyses and identification of oil compounds were performed as previously described (2).

Antibacterial testing
The activity of the essential oil samples was tested towards thirteen model bacteria. The inocula of the bacterial strains were prepared from overnight broth cultures and the suspensions were adjusted to 0.5 McFarland standard turbidity (corresponding to 108 CFU/mL) depending on genera-consensus standard by the Clinical and Laboratory Standards Institute (8).

Micro-well Dilution Assay and Microdilution Checkerboard Assay
Micro-well dilution assay and microdilution checkerboard assay were used to determine the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and to assess antimicrobial combinations in vitro, as previously described (2).

Chemical composition of the essential oil
The yield of P. longifolium essential oil was 0.31% (w/w). Based on GC and GC-MS analysis of P. longifolium oil, 59 components were identified that represented 95.40% of the total detected constituents ( Table 1). The monoterpene hydrocarbons were the most abundant compound class in the oil (61.60%), and they were dominated by myrcene (15.88%), α-phellandrene (11.28%) and limonene (8.23%).

Antibacterial activity
The essential oils were tested for their antibacterial activity by the broth microdilution method to determine the MIC and MBC values against thirteen model bacteria ( Table 2). The results from the antibacterial assay showed that P. longifolium essential oil possessed slight antimicrobial activities, with MIC and MBC values in the range from 2427.2 to 38835.2µg/ml. Grampositive bacteria were generally found to be more sensitive than gram-negative ones. The reference antibiotics were active in the following ranges of concentration: tetracycline, 0.5 to 1024.0 µg/ml; streptomycin, 0.5 to 256.0 µg/ml and chloramphenicol, 1.0 to 2048.0µg/ml.

Interaction between the essential oil and antibiotics
The results of the possible interactions between the essential oil and the antibiotics are given in Table 3. From 135 tested combinations between P. longifolium oil and the three antibiotics, 50 (37.04%) showed synergism, while 29 (21.48%) had an additive and 56 (41.48%) had an antagonistic effect. The best antibacterial activities were obtained with the combination of P. longifolium oil and chloramphenicol. For this combination, a synergistic effect of 66.67% was recorded (FIC indices in the range 0.21-0.87). The highest percentage of the additive effect (26.67%) was registered again in the oil/chloramphenicol combination, while the highest percentage of the antagonistic effect (77.78%) was noted for the oil/tetracycline association. The maximum effect combination of essential oil and antibiotics was expressed toward K. pneumoniae ATCC 700603. The results of the checkerboard assay showed a synergistic effect of 51.85%, an additive effect of 22.22% and an antagonistic effect of 25.93% for this bacterial strain.

Discussion
The composition of essential oil isolated from the aerial parts of P. longifolium was found similar in comparison to the composition of the essential oil of P. longifolium from Orjen, Montenegro. Both essential oils accumulate monoterpene hydrocarbons, as the dominant class of compounds (9).
The antibacterial activity of P. longifolium essential oil displayed significant variation among the different bacteria species but remained much lower than the activities of the standard antibiotics. In research of the antibacterial activity of Peucedanum membranacea Boiss. against four bacteria, inhibitory values in the range 32-250 µg/mL were found (6).
The interaction of essential oils with antibiotics is one of the novel ways to overcome bacterial resistance. The presented results of total effects of essential oil/antibiotic combinations fully justified the purpose of the study and indicated an encouraging fact, i.e., an essential oil with low antibacterial activity may exhibit synergistic and additive effects in association with conventional antibiotics. The combination of P. longifolium oil 21    and tetracycline exhibited a predominantly antagonistic effect. A very strong antagonistic effect was registered against S. aureus ATCC 29213. It should be mentioned that the essential oil in these combinations had no effect on the decreasing of the MIC values of tetracycline, except for P. mirabilis ATCC 12453 and P. aeruginosa ATCC 27853 (the MIC value of tetracycline decreased up to 2-fold). The explanation of the mechanism of interactions that produce antagonistic effects has been less studied. Hypo-theses, which are discussed in some studies, included the use of antibacterial compounds (or mixtures) that act on the same site of the bacteria, and chemical interactions among the antibacterial compounds (10). The combination of P. longifolium oil and streptomycin against K. pneumoniae ATCC 700603 and P. mirabilis ATCC 12453 exhibited a strong synergistic effect and decreased the MIC value of streptomycin 10-fold. In contrast, the oil-streptomycin association against E. coli ATCC 25922 and P. aeruginosa ATCC 27853 performed a strong antagonistic effect and had no effect on the decreasing of the MIC values of streptomycin. Elucidation of the mechanism of antibacterial action based on the interaction of essential oil/antibiotic is not simple. All interactions between antibacterial compounds could change the effectiveness and relationships (synergistic, additive or antagonistic) in competition for the pos-sible primary target. On the other hand, a synergistic multitarget effect could occur by involving proteins, enzymes, ribosomes, nucleic acids, receptors and ion channels (11).
The combination of P. longifolium oil and chloramphenicol against all the tested bacteria exhibited a predominantly synergistic effect and decreased the MIC value of chloramphenicol 10fold, for all the tested bacterial strains, except for P. mirabilis ATCC 12453 (the MIC value of chloramphenicol decreased 3.3-fold). There are some generally accepted mechanisms of antibacterial interaction that produce synergism, including inhibition of protective enzymes, combination of membrane active agents, sequential inhibition of common biochemical pathways and the use of membranotropic agents to enhance the diffusion of other antimicrobials (12). Based on the present results, it could be hypothesized that the antibacterial activity and hence the synergistic effect of P. longifolium essential oil is connected with the high percentage of hydrocarbons, the most abundant compound class.
The antibacterial activity of hydrocarbons can be explained by the interactions of these compounds with the membrane and with membrane constituents of bacteria. As a result of accumulated lipophilic molecules, the membrane loses its integrity, and an increase in permeability to protons and ions can be observed. In addition, it has been found that proteins embedded in the membrane are also affected (13). All these considerations prompted the hypothesis that the components of P. longifolium essential oil, with the hydrocarbons as the major compounds, favor the mechanism of action of chloramphenicol, the main effect of which is the inhibition of the bacterial enzyme peptidyl transferase, thereby preventing the growth of the polypeptide chain during protein synthesis (14). The obtained results indicate that chloramphenicol, not currently used as a therapeutic agent against gram-negative bacteria, in combination with an appropriate essential oil has significant antibacterial activity, especially against gram-negative bacteria. Moreover, its minimum effective dose is signifi-cantly reduced, and consequently possible toxic side effects are decreased. In addition, it can be assumed that in research of the antibacterial effects of essential oil-antibiotic combinations, the choice of gramnegative or gram-positive bacterial species is not decisively significant. In other words, the proper essential oil-antibiotic associa-tion will act equally stronger or weaker against all gram-positive and gram-negative bacterial strains.

Conclusion
In the present study, the chemical composition of P. longifolium essential oil was examined and a correlation among the anti-bacterial activities of the essential oil-antibiotic combinations was realized. It was shown that monoterpene hydrocarbons, with myrcene (15.88%) as the dominant constituent, were the most abundant compound class of the essential oil of P. longifolium from Serbia. The researched essential oil exhibited slight antibacterial activity against the tested bacterial strains in vitro. On the contrary, essential oil of P. longifolium possesses significant synergistic potential in combination with streptomycin and chloramphenicol (FIC indices in the range 0.21-0.87). These combinations reduced the minimum effective dose of the antibiotics and, consequently, minimized their adverse side effects. In addition, investigated interactions are especially successful against gram-negative bacteria, the pharmacological treatment of which is very difficult nowadays.