Central European Journal of Biology

* E-mail: sanda@pharma.hr

Research Article

1Department of Pharmacognosy, Faculty of Pharmacy and Biochemistry, University of Zagreb, HR-10000 Zagreb, Croatia

2Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, HR-10000 Zagreb, Croatia

3Food Control Center, Faculty of Food Technology and Biotechnology, University of Zagreb, HR-10000 Zagreb, Croatia

4Kemika, Zagreb, HR-10000, Croatia; Present address: Galapagos Research Center, HR-10000 Zagreb, Croatia

5Institute for Public Health “Dr. Andrija Štampar”, HR-10000 Zagreb, Croatia

Sanda Vladimir-Knežević1,*, Ivan Kosalec2, Marija Babac1, Marinko Petrović3, Jovica Ralić4, Biserka Matica5, Biljana Blažeković1

Antimicrobial activity of Thymus longicaulis C. Presl essential oil against respiratory pathogens

1. IntroductionEssential oils produced by aromatic plants have been used from ancient times as antiseptics and anti-infectious agents. Since the discovery and development of chemotherapeutic agents, especially antibiotics, the use of essential oils has been reduced, but such oils have been used traditionally for the prevention and therapy of respiratory tract infections [1]. Respiratory infections are the most frequent reason for primary health care consultation. Epidemiological studies indicated

that one fourth to one third of patients visiting general practitioners suffer from acute respiratory infections. Paediatric respiratory tract infections are especially associated with significant morbidity and mortality. Additionally, antimicrobial resistance among respiratory tract pathogens has become an increasing problem world-wide [2,3]. Since infections with respiratory tract pathogens are common in all age groups, their contribution to the total consumption of antimicrobial agents in a population is therefore significant. Despite the many infectious disease control and surveillance

Cent. Eur. J. Biol. • 7(6) • 2012 • 1109-1115DOI: 10.2478/s11535-012-0088-2

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Received 14 March 2012; Accepted 28 July 2012

Keywords: Thymus longicaulis • Essential oil • Thymol • Antimicrobial activity • Respiratory pathogens

Abstract: Thymus longicaulis C. Presl is a small aromatic perennial herb used as a traditional remedy for cold, flu and cough. Composition of the essential oil of T. longicaulis from Croatia and its in vitro antimicrobial activity against the most common respiratory pathogens were evaluated. The yield of essential oil obtained by hydrodistillation from aerial plant parts was 1.2%. According to the GC-MS analysis, a total of forty one compounds (99%) were identified. Thymol (46.3%), γ-terpinene (16.2%), thymyl methyl ether (11.4%), and p-cymene (9.4%) were the main components. Antimicrobial activity of the essential oil against six clinically isolated bacterial and yeast strains was determined using standard disc agar diffusion method and microdilution broth assay. The essential oil exhibited antimicrobial activity towards all tested respiratory pathogens. The most sensitive strains were Haemophilus influenzae and Streptococcus pneumoniae(MIC=0.78 mg/mL),while Staphylococcus aureus was the most resistant (MIC>25.00 mg/mL). Our results indicate that T. longicaulis essential oil could be effective against clinically relevant respiratory pathogens which have the ability to develop resistance to antimicrobial drugs.

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Antimicrobial activity of Thymus longicaulis C. Presl essential oil against respiratory pathogens

programs, the medical community has not been able to stop the global spread of resistant microorganisms. The increasing antimicrobial resistance in major respiratory pathogens such as Streptococcus pneumoniae, Staphylococcus aureus and Gram-negative bacteria has severely restricted the treatment options. Respiratory infections caused by multidrug-resistant bacteria are associated with a greater likelihood of inappropriate antimicrobial therapy and poor clinical outcome [4,5]. The quest for a solution to the global problem of antibiotic resistance has often focused on finding new antimicrobial compounds from a variety of natural sources. In this regard, plant essential oils may offer a great potential for the development of novel antibacterial therapies and complementary treatments.

The genus Thymus (Lamiaceae) is noteworthy for the numerous wild-growing plants biosynthesizing a remarkable amount of volatile compounds. Thymus species are perennial, aromatic herbs and subshrubs native to Europe and North Africa [6]. They are commonly used as culinary herbs, flavouring agents and medicinal plants. Recent studies have shown strong antibacterial, antifungal, antiviral, antiparasitic, spasmolytic and antioxidant activities of Thymus species. It has been considered that a part of these effects is due to the volatile constituents [7-13]. Therefore, there is a considerable research interest in the chemical composition analysis of Thymus essential oils and their biological activities.

Thymus longicaulis C. Presl is a small aromatic perennial herb and a typical representative of the Illyric-Mediterranean flora. A great number of subspecies, varieties and forms were described within this polymorphic species [14]. It is a traditional remedy for cold, flu, cough, nephritis and abdominal pain [15-18]. To the best of our knowledge, there are several reports on the essential oil analysis of different taxa of T. longicaulis, and the results revealed their marked chemical variations depending on genetically determined properties, localities and environmental factors [19-26]. Three reports have been published concerning the antimicrobial activity of T. longicaulis recently. Essential oils isolated from T. longicaulis of Italian origin as well as from its two subspecies chaubardi and longicaulis growing in Turkey showed activities against several standard strains of common pathogenic bacteria and Candida albicans [27,28]. Susceptibility of foodborne bacteria on T. longicaulis essential oil was also assessed [29].

To find new antimicrobial resources, the present study aims to evaluate the composition of the essential oil of T. longicaulis growing in Croatia and its in vitro antimicrobial activity against major respiratory tract pathogens that show increasing resistance to commonly

prescribed antimicrobials. Although this group of microorganisms has shown a high susceptibility to several other essential oils [1,30], activity of T. longicaulis essential oil against clinically relevant respiratory tract pathogens has not been investigated thus far.

2. Experimental Procedures2.1 Plant material and essential oil isolationAerial parts of wild-growing Thymus longicaulis C. Presl were collected at the flowering stage from Jasenice (Central Dalmatia, Croatia, 130 m a.s.l.) in May 2010. The plant sample was authenticated by Department of Botany and Botanical Garden, Faculty of Science, University of Zagreb, Croatia, where a voucher specimen of the plant is deposited (No. 819-4). The aerial parts of the plant were dried at room temperature, then crushed and hydrodistilled for 3 hours using Clevenger-type apparatus. The oil was dried over anhydrous sodium sulphate and stored at 4°C [31].

2.2 Gas Chromatography/Mass Spectrometry analysis (GC/MS)

The essential oil was analysed by GC/MS on CP-3800 gas chromatograph coupled with Saturn 2000 mass selective detector (Varian, Palo Alto, USA), equipped with CP Sil-8 capillary column (30 m x 0.25 mm, film thickness 0.25 μm). Operating conditions were as follows: carrier gas, helium at a flow rate of 1 mL/min; column temperature, 60-250°C at a rate of 4°C/min; injector temperature, 280°C; injected volume 0.1 μL; split ratio, 1:100. Mass spectra were recorded at 70 eV and were scanned in the range 45-350 uma. The individual components of the essential oil were identified by comparison of their retention indices with either those of the literature [32] or with those of authentic compounds available in our laboratory. The retention indices were determined in relation to a homologous series of n-alkanes (C8-C20) under the same operating conditions. Further identification was made by comparison of their mass spectra with either those stored in NIST2000 and in-house library or with mass spectra from the literature [33,34]. Relative amounts of the essential oil components were obtained by peak area normalization.

2.3 Antimicrobial assaysActivity of T. longicaulis essential oil against respiratory pathogens was evaluated by disc diffusion method and microdilution broth susceptibility assay. Fresh clinical bacterial (Haemophilus influenzae, Neisseria meningitidis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and yeast

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(Candida albicans) isolates used in this study were obtained from patients with clinical manifested diseases of the upper respiratory tract, and deposited in Collection of Microorganisms at the Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb. All tested bacterial and fungal strains were maintained on an agar slant and stored at 4°C. Bacteriological media used for culture and multiplication tested microorganisms was Müller-Hinton agar for bacteria or Sabouraud 2%-glucose agar for fungi. Inoculum of each strain to be tested was prepared with fresh cultures by suspending the microorganisms in sterile saline and adjusting the density to 0.5 McFarland standard (1.5x108 CFU/mL)using nephelometer (bioMérieux, France). In the first step, antimicrobial activity of the essential oil in question was studied by the disc diffusion method [35]. Amoxicilline-clavulanate (20 μg/10 μg) and fluconazole (25 μg) discs (AB Biodisk, Sweden) were used in order to control the sensitivity of tested clinically isolated bacterial and yeast strains, respectively. Sterile paper discs, 6 mm in diameter (Fluka, Switzerland), were soaked with 10 μL of the oil and placed on the surface of inoculated agar plates (Müller-Hinton agar for bacteria or Sabouraud 2%-glucose agar for fungi). After maintaining at 4°C for 2 h, the plates were incubated under aerobic conditions at 37°C for 24 h (bacteria) and 25°C for 48 h (fungi). The diameters of the inhibition zones were measured in millimetres. All tests were carried out in quadruplicate and the results are expressed as mean ± standard deviation. Microdilution broth susceptibility assays were employed for determination of minimum inhibitory concentrations (MIC) of the essential oil [36]. Amoxicilline-clavulanate and fluconazole served as reference antimicrobials. Stock solution of the essential oil was prepared in dimethylsulfoxide. Serial twofold dilutions of the essential oil (0.20-25.00 mg/mL) in volumes of 100 μL were prepared in the liquid nutrient medium, Müller-Hinton broth (Fluka, Germany) for bacteria and RPMI

1640 with 2% glucose (Sigma-Aldrich, USA) for fungi, in 96-well microtiter plates (Kartell, Italy). Aliquots of 10 μL of diluted microbial suspension were then added to each well (5x105 CFU/mL). After incubation at 37°C for 24 h (bacteria) and 25°C for 48 h (fungi), the growth of microorganisms was indicated by the presence of turbidity. To establish the MIC, 10 μL of broth was removed from each well and subcultured on the adequate agar plate. The MIC was recorded as the lowest concentration of the agent that resulted in a significant decrease in inoculum viability (>80%). Each treatment was performed in quadruplicate and the results are expressed as mean ± standard deviation.

3. Results and DiscussionThe yield of essential oil obtained by hydrodistillation from dried aerial parts of T. longicaulis was 1.2% (V/m). According to the GC/MS analysis, forty one compounds were identified, representing 99% of the total oil. As indicated in the Table 1, the essential oil was characterized by a high percentage of the monoterpene fraction (95.6%), dominated by oxygenated monoterpenes (65.5%) and hydrocarbons (30.1%). Sesquiterpenes constituted only 3.4% of the oil. Among reported components, thymol (46.3%), γ-terpinene (16.2%), thymyl methyl ether (11.4%) and p-cymene (9.4%) were identified as the most abundant compounds (Figure 1), followed by borneol, β-caryophyllene, δ-3-carene and linalool ranged from 1.4% to 2.2%. These results, obtained for the plant sample originating from the Central Dalmatia region of Croatia, are in partial agreement with previously reported data on T. longicaulis collected in the other Croatian geographic region (Istria) in which thymol and p-cymene were the predominant components [19]. On the other hand, our findings differ from previous data on the essential oil of T. longicaulis from Serbia and Italy which demonstrated α-terpinyl

Thymol

Thymyl methyl ether

γ-Terpinene

p-Cymene

Figure 1. The main components of the essential oil of Thymus longicaulis C. Presl.

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acetate [24], p-cymene [25] and geraniol [28] as the main components of these oils.

Antibacterial and antifungal activities of the essential oil isolated from T. longicaulis against pathogens of the respiratory tract were assessed according to the inhibition zones (ZI) as well as values of minimum inhibitory concentrations (MIC) and the results are presented in Table 2. The essential oil was effective against all tested microorganisms (ZI, 34-53 mm), generating the largest inhibition zones for Neisseria meningitidis. A moderate to strong antimicrobial activity of the investigated essential oil was also confirmed using microdilution broth assay (Table 2). No obvious difference in susceptibility was found among Gram-positive and Gram-negative bacteria tested. Haemophilus influenzae and Streptococcus pneumoniae were the most susceptible to the essential oil (MIC, 0.78 mg/mL), followed by Streptococcus pyogenes, Neisseria meningitidis and Candida albicans (MIC, 2.74-12.50 mg/mL), while Staphylococcus aureus was the most resistant (MIC>25.00 mg/mL). Kunduhoglu et al. [37] recently reported that ethanolic extract of T. longicaulis from Turkey also possesses antimicrobial activity against several bacterial strains including S. aureus.

Though the activity of T. longicaulis essential oil against tested microorganisms was of varying intensity and much less in comparison with reference antimicrobials, the results clearly demonstrated that the essential oil in question has a significant antimicrobial capacity and acts toward bacterial and fungal respiratory pathogens. Antimicrobial activity of the essential oils would be expected to relate to the respective composition and structural configuration of the constituent components of the oils. The components with phenolic structure are known to be highly active antimicrobial agents [38]. Thymol, a main constituent of various Thymus essential oils, was recognised to possess a wide spectrum of antibacterial and antifungal activity. Guo et al. [39] proved the thymol effectiveness against clinical isolates of fluconazole-sensitive and fluconazole-resistant Candida albicans. Thymol also showed a strong antibacterial activity and was considerably more toxic against S. aureus than carvacrol, γ-terpinene and p-cymene [40]. Therefore, the antimicrobial effect of T. longicaulis essential oil demonstrated in this paper is most probably due to the presence of thymol, which constituted almost half of the total oil content. Even though biological properties of an essential oil are mainly attributed to its major components, some studies revealed that minor components as well as possible synergistic or antagonistic effects between the substances may also affect the antimicrobial activity [41,42].

Compound RIa Percentage

Tricyclene 922 tr

α-Thujene 925 0.6

α-Pinene 933 0.4

Camphene 947 0.6

1-Octen-3-ol 963 0.3

3-Octanone 966 0.5

β-Pinene 973 0.1

3-Octanol 980 0.1

Myrcene 983 0.6

α-Phellandrene 996 0.1

δ-2-Carene 1004 tr

δ-3-Carene 1009 1.6

p-Cymene 1012 9.4

1,8-Cineole+Limonene 1021 0.4

cis-β-Ocimene 1039 tr

γ-Terpinene 1051 16.2

p-Menth-3-en-1-ol 1055 0.2

p-Cymenene 1073 tr

Terpinolene 1079 0.1

Linalool 1083 1.4

Sabinene hydrate 1105 tr

Camphor 1120 0.1

Borneol 1150 2.2

p-Cymen-8-ol 1160 tr

Terpinen-4-ol 1163 0.5

α-Terpineol 1173 0.1

Thymyl methyl ether 1215 11.4

Carvacryl methyl ether 1226 0.9

Thymol 1268 46.3

Carvacrol 1275 1.4

Thymyl acetate 1324 0.1

β-Burbonene 1382 tr

β-Caryophyllene 1416 2.1

Bicyclosesquiphellandrene 1425 tr

α-Humulene 1449 0.2

γ-Gurjunene 1474 0.2

α-Himachalene 1498 0.6

γ-Cadinene 1503 tr

β-Cadinene 1512 0.1

Spatulenol 1562 0.1

Caryophyllene oxide 1568 0.1

Total 99.0

Table 1. Chemical composition of the essential oil of Thymus longicaulis C. Presl.

a Retention index relative to n-alkanes on CP Sil-8 column;

tr - traces (<0.05%)

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[1] Inouye S., Yamaguchi H., Takizawa T., Screening of the antibacterial effects of a variety of essential oils on respiratory tract pathogens, using a modified dilution assay method, J. Infect. Chemother., 2001, 7, 251-254

[2] Baquero F., Barrett J.F., Courvalin P., Morrissey I., Piddock L., Novick W.J., Epidemiology and mechanisms of resistance among respiratory tract pathogens, Clin. Microbiol. Infect., 1998, 4, 2S19-2S26

[3] Mlynarczyk G., Mlynarczyk A., Jeljaszewicz J., Epidemiological aspects of antibiotic resistance in respiratory pathogens, Int. J. Antimicrob. Agents, 2001, 18, 497-502

[4] Kristiansen B.E., Sandnes R.A., Mortensen L., Tveten Y., Vorland L., The prevalence of antibiotic resistance in bacterial respiratory pathogens from Norway is low, Clin. Microbiol. Infect., 2001, 7, 682-687

[5] Song J.H., Chung D.R., Respiratory infections due to drug-resistant bacteria, Infect. Dis. Clin. North Am., 2010, 24, 639-653

[6] Morales R., Synopsis of the genus Thymus L. in the Mediterranean area, Lagascalia, 1997, 19, 249-262

[7] Stahl-Biskup E., Essential oil chemistry of the genus Thymus - a global view, In: Stahl-Biskup E., Saez F. (Eds.), Thyme: The genus Thymus, Taylor and Francis, London, 2002

[8] Astani A., Reichling J., Schnitzler P., Comparative study on the antiviral activity of selected monoterpenes derived from essential oils, Phytother. Res., 2010, 24, 673-679

[9] Dahbi A., Bellete B., Flori P., Hssaine A., Elhachimi Y., Raberin H., et al., The effect of essential oils from Thymus broussonetii Boiss on transmission of Toxoplasma gondii cysts in mice, Parasitol. Res., 2010, 107, 55-58

[10] Engelbertz J., Lechtenberg M., Studt L., Hensel A., Verspohl E.J., Bioassay-guided fractionation of a thymol-deprived hydrophilic thyme extract and its antispasmodic effect, J. Ethnopharmacol., 2012, 141, 848-853

[11] Fadli M., Saad A., Sayadi S., Chevalier J., Mezrioui N.E., Pagès J.M., et al., Antibacterial activity of

Inhibition zone [mm]a Minimum inhibitory concentration [mg/mL]

Microorganisms Essential oilb Amoxicilline-clavulanatec Fluconazoled Essential oil Amoxicilline-

clavulanate (×10 -3)Fluconazole

(×10 -3)

Haemophilus influenzae 42±2 24±1 - 0.78±0 0.44±0.13 -

Neisseria meningitidis 53±2 23±0 - 5.47±1.56 0.02±0.01 -

Staphylococcus aureus 35±1 29±1 - >25.00 0.03±0 -

Streptococcus pneumoniae 43±1 25±0 - 0.78±0 0.02±0 -

Streptococcus pyogenes 41±2 42±2 - 2.74±0.78 0.03±0.01 -

Candida albicans 34±0 - 30±1 12.50±0 - 0.15±0

Table 2. Antimicrobial activity of Thymus longicaulis C. Presl essential oil against respiratory pathogens.

Results are expressed as mean ± standard deviation. a Includes diameter of disc (6 mm); b 10 μL/disc; c (20 μg/10 μg)/disc; d 25 μg/disc; - not tested

4. ConclusionsIn conclusion, our results clearly indicate that essential oil of T. longicaulis originating from Croatia belongs to a thymol chemotype, and provide additional evidence of intraspecific chemical variability of this plant species. The results of antimicrobial activity obtained by two different in vitro assays show that the essential oil examined could be effective against clinically relevant respiratory pathogens. These findings also support the ethnopharmacological use of T. longicaulis and

encourage further research to assess its value in modern phytotherapy.

Acknowledgements This research was funded by the Ministry of Science, Education and Sports of Republic of Croatia (Project number: 006-0061117-1238). The authors would like to thank Miško Plazibat, PhD for autentification of plant material.

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