Antimicrobial activity of essential oils: the possibilities of TLC–bioautography

Authors


  • This paper was published in Flavour and Fragrance Journal as part of the special issue based on the lectures given at the 40th International Symposium on Essential Oils, held at Savigliano, Italy, 6–9 September 2009, organized by Carlo Bicchi and Patrizia Rubiolo

Abstract

Essential oils are well-known for their antimicrobial activity against different plant and human pathogenic microorganisms. The results of the most commonly used antimicrobial assays are very different; sometimes their reliability is questionable, therefore standardized methods need to be used to solve this problem. The present study aims at the phytochemical characterization of some essential oils (thyme, lavender, eucalyptus, spearmint and cinnamon) that are important from the therapeutic and economic aspects and the optimized microbiological investigation of the effect of essential oils on human and plant pathogenic microorganisms. The chemical composition of the essential oils was analysed with thin-layer chromatography (TLC) and their composition was controlled by gas chromatography (GC). The antibacterial effect was investigated using the TLC-bioautographic method. The solvents applied in TLC developing systems were also tested. Our results showed that toluene, ethyl acetate, ethanol and chloroform as solvents used in the assay had no inhibiting effect on the test bacteria. The antibacterial activity of thyme, lavender and cinnamon oils and their main components (thymol, carvacrol, linalool, eugenol) was observed in the case of two plant pathogenic bacteria (Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. phaseolicola) and some human pathogens (Staphylococcus epidermidis, S. saprophyticus and two strains of S. aureus, including one methicillin-resistant strain). On the whole, the antibacterial activity of essential oils can be related to their most abundant components, but the effect of the minor components should also be taken into consideration. Direct bioautography is more cost-effective and compares better with traditional microbiological laboratory methods (e.g. disc-diffusion, agar-plate technique). Copyright © 2010 John Wiley & Sons, Ltd.

Introduction

Essential oils are used in a variety of fields, such as pharmacology, medical microbiology, phytopathology and food preservation. The number of studies focusing on these substances, as well as their application as new potential antibiotic agents against plant and human microorganisms, has recently increased.[1–3] These experiments are important because antibiotics or chemical sprays applied for prevention and treatment in agriculture and human medicine can unfortunately cause selective pressure leading to the spread of resistant mutants.

Previous studies on the antimicrobial activity of essential oils in vitro described a wide range of assays with different parameters (agar recipes, incubation time, solvents, microorganisms),[4–6] so the results from the assays are very different and sometimes their reliability is questionable. The most widely used methods applied include disc diffusion, agar absorption, agar dilution and broth dilution assays. Both the diffusion and dilution methods have been widely and routinely used for many years in antibacterial susceptibility testing in clinical microbiology.[7] It is important to note that the substances tested with these methods are generally hydrophilic (e.g. antibiotics), so the tests have been optimized to this condition.

Essential oils are volatile, complex and viscous substances that are insoluble in water, so the common screening methods mentioned above are inadequate for their antimicrobial testing. Therefore, there is a need for an optimized and reproducible assay for assessing the antibacterial effect of these oils.

Direct bioautography combined with thin layer chromatographic (TLC) separation is a rapid and sensitive screening method for the detection of antimicrobial compounds. Test microorganism cultures are capable of growing directly on the TLC plate, so each step of the assay is performed on the sorbent. Similarly to the common antimicrobial screening methods, TLC-bioautography must be carried out under controlled conditions, since the experimental conditions (e.g. solvent, sampleapplication, resolution of compounds, type of microorganism, incubation time, etc.) may influence the result.[8] Paper chromatography and bioautographic detection of antibiotics were performed for the first time in 1946.[9] Fifteen years later, Fischer and Lautner used TLC for this purpose.[10] The advantages of direct bioautography are that it is suitable for evaluating complex plant extracts and facilitates rapid, economic and easy evaluation. The use of bioautography to detect antimicrobial compounds effective against plant and human pathogenic bacteria has been reported in the literature.[11–13]

The present study aimed at the phytochemical characterization of the essential oil of thyme, lavender, eucalyptus, spearmint and cinnamon by TLC and the detection of antibacterial activity of essential oils and their main components against some human and plant pathogenic bacteria. These essential oils are important from therapeutic as well as economic viewpoints and their antibacterial effect has already been proved in human medicine. The composition of essential oils was checked using gas chromatography. In order to optimize the bioautographic method, the effect of various solvents applied in TLC developing systems were also studied. To the best of our knowledge, the present study is the first to investigate the biological activity of the above essential oils against Xanthomonas, Pseudomonas and Staphylococcus strains using direct bioautographic assay.

Experimental

Essential Oil Samples

The essential oils of thyme (Thymus vulgaris L.), lavender (Lavandula angustifolia Mill.), eucalyptus (Eucalytus globulus Labill.), spearmint (Mentha spicata L.) and cinnamon (Cinnamomum zeylanicum Presl.) were obtained from a Hungarian pharmacy (Herbaria, Hungary). The quality of the essential oils used in the present study met the standards described in the European Pharmacopoeia, 4th edn.

Gas Chromatography

The five essential oils were analysed using a Fisons GC 8000 gas chromatograph (Carlo Erba, Milan, Italy), equipped with a flame ionization detector (FID). An Rt-β-DEXm (Restek) capillary column, 30 m × 0.25 mm i.d., 0.25 µm film thickness, was used. The carrier gas was nitrogen at 6.86 ml/min flow rate; 0.2 µl of a 0.25% solution was injected (5 µl essential oil in 2 ml chloroform). Splitless injection was made. The temperatures of the injector and detector were 210°C and 240°C, respectively. The oven temperature was increased at a rate of 8°C/min from 60°C to 230°C, with a final isotherm at 230°C for 5 min. Percentage evaluation of compounds was carried out by area normalization; identification of peaks were made by comparison of retention times of standards and co-addition of standards. All measurements were made in duplicate.

This analysis was meant to check only the main components and the composition of essential oils examined in this study.

Direct Bioautography

This process can be divided into three parts: cultivation of test bacteria for dipping; planar chromatographic separation and detection; and treatment for post-chromatographic detection.

Cultivation of test bacteria for dipping.

Plant pathogenic test organisms were: Pseudomonas syringae pv. phaseolicola (Burkholder), which was isolated from an infected bean sample; and Xanthomonas campestris pv. vesicatoria (Doidge) Dye, which was isolated from infected tomato and green pepper samples. These bacteria were obtained from the Bacteriological Laboratory of Health and Soil Conservation Service of Baranya County, Hungary.

Human pathogenic test bacteria were: strains of Staphylococcus epidermidis, S. saprophyticus, S. aureus (HNCMB 112002) and methicillin-resistant S. aureus, which were isolated from clinical samples and obtained from the Institute of Medical Microbiology and Immunology, University of Pécs, Hungary. The test organisms were maintained on Mueller–Hinton agar (Oxoid, UK).

For bioautography, bacteria were grown in 100 ml Mueller–Hinton nutrient broth, pH 7.3, at 28°C and 37°C (in the case of plant and human pathogenic bacteria, respectively) in a shaker incubator at a speed of 60 rpm for 24 h. The bacterial suspension was diluted with fresh nutrient broth to OD600 = 0.4, which corresponds to approximately 4 × 107 colony-forming units (cfu)/ml.

Planar chromatographic separation and detection.

Chromatography was performed on 10 × 20 cm silica gel 60 F254 aluminium sheet TLC plates (Merck, Germany). Before use, the plates were preconditioned by heating at 120°C for 3 h.

The antibacterial activity of the main components (thymol, carvacrol, eugenol, carvone, linalool, 1,8-cineole, borneol) of the essential oils examined in this study was investigated with direct bioautography. Pure samples of these chemicals were obtained from Sigma (St. Louis, MO, USA), dissolved in ethanol to give solutions containing 5 µl/ml, and 4 µl (equivalent to 0.02 µl undiluted standard) was applied to the plates, using Minicaps capillary pipettes (West Germany). For the solution of the essential oils 20 µl was dissolved in 1 ml ethanol, and 4 µl of it (equivalent to 0.08 µl undiluted oil) was applied to the TLC plate with Minicaps capillary pipettes. The position of the starting line was 1.5 cm from the bottom and 1.5 cm from the left side. The standards of the main components of the essential oils were applied to the TLC plates next to the spots of the oils. After the sample application, the TLC plates were developed with the previously optimized mobile phase. For the separation of essential oils, toluene–ethyl acetate, 93:7, as a mobile phase is recommended.[14] The development mode was ascendant chromatography in a saturated twin-trough chamber (Camag, Switzerland). All TLC separations were performed at room temperature (20°C). For the bioautographic assay, solvents applied in TLC developing systems were also tested.

After chromatographic separation, the absorbent layers were dried in an oven at 90°C for 5 min to remove the solvent completely. Alcoholic vanillin–sulphuric acid reagent[14] was used to visualize the separated compounds. The developed layers were dipped into this reagent and heated for 5 min at 100°C. Detection of the separated compounds was performed according to Rf value and colour of the standards (vis). Although the TLC plate was fluorescent, the evaluation of the separated compounds by UV was not done. At UV 365 nm no characteristic fluorescence of terpenoids and propylphenols was noticed.[14] It should be noted that the TLC plate for bioautography was processed in parallel without final development with reagent, because this does not affect the success of the subsequent microbial detection process in bioautography.

Post-Chromatographic Detection

For bioautography the plates developed were dipped for 10 s in approximately 50 ml culture medium containing inocula and then dried under an air flow for 2 min. The purity of the culture was checked by cultivation on Mueller–Hinton agar plates. TLC plates were incubated in a water-vapour chamber (dimensions: 20 × 14.5 × 5 cm) at 28°C and 37°C (in the case of plant pathogenic and human pathogenic bacteria, respectively) for 17 h, then dipped for 10 s in an aqueous solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma; 0.1 g/60 ml). The treated plates were incubated at 28°C for 2 h and 37°C for 4 h, respectively, then dipped in 70% ethanol for 10 s and dried at room temperature. The inhibition zones of the separated compounds were visualized by detecting dehydrogenase activity with tetrazolium salt-based reagents. On the TLC plate metabolically active bacteria convert the tetrazolium salt, MTT, into formazan dye. Thus, the inhibition zones were visible as pale spots against a dark blue background. The diameters of zones of inhibition were measured in mm. All measurements were performed in triplicate. The TLC plates were photographed with a Canon PowerShot A95 camera.

Results and Discussion

Gas Chromatographic Detection

The essential oil composition of thyme, lavender, eucalyptus, spearmint and cinnamon was determined by gas chromatography. With this analysis we wanted to check only the main components of these oils. The components were identified by comparing their retention times and relative retention factors with those of authentic standards and oils of known composition, and by peak enrichment. Evaluation of percentage composition was based on peak areas, by area normalization, on the basis of two parallel measurements.

Thymol (41.3%) was the main component in the essential oil of thyme. Linalyl acetate (44.1%) and linalool (40.4%) were the main components in the oil of lavender. Carvone (70.4%), 1,8-cineole (90.0%) and trans-cinnamic aldehyde (78.5%) were the main compounds in the oil of spearmint, eucalyptus and cinnamon, respectively. Among the other constituents, p-cymene, β-caryophyllene, limonene, carvacrol and camphor were also characteristic of the oils, but smaller amounts were present. Although Rt-β-DEXm is a chiral GC phase, the enantiomers of carvone and linalyl acetate were not resolved.

TLC

Bioautography of the five essential oils was performed by parallel analysis on 10 × 20 cm TLC plates. Lanes were duplicated on the left and right halves of a single plate. After the development the plates were cut into two halves. One half was dipped into the alcoholic vanillin–sulphuric acid reagent to detect the essential oil components, while the other was dipped into the bacterial suspension for the bioautographic procedure. Thus, the chromatographic conditions were the same for both. In the oil of thyme, thymol and carvacrol could be identified as a single red zone at Rf = 0.57. The linalool reference was identified as a blue zone at Rf = 0.39. According to gas chromatographic analysis, cinnamic aldehyde (78.5%) was the main component and the amount of eugenol was only 2.7% in the cinnamon oil. However, the two compounds could not be separated by TLC development with toluene–ethyl acetate (93 + 7) mixture, due to their similar Rf values. Cinnamic aldehyde was only identified by reference to Wagner,[14] while eugenol was determined as a brown zone at Rf = 0.53 on the basis of co-chromatography with eugenol standard. Since the quantitative difference between thymol (41.3%) and carvacrol (3.2%), as well as cinnamic aldehyde (78.5%) and eugenol (2.7%) was so large in thyme and cinnamon oil, respectively, it was not worth trying to separate them on TLC plate. Instead, we applied standards of these compounds to the plate as spots, for comparison. In the oil of eucalyptus the main component was 1,8-cineole (blue zone, Rf = 0.48) and in the oil of lavender linalyl acetate was identified by reference to Wagner[14] as the main component (blue zone, Rf = 0.65). Linalool was also identified as a blue zone at Rf = 0.39 in the oil of lavender. Carvone could be identified as a red-violet zone at Rf = 0.53 in the spearmint oil (Figure 1).

Figure 1.

TLC separation of the essential oil of eucalyptus, lavender and spearmint on silica gel 60 F254. Solvent, toluene–ethyl acetate, 93 + 7. Detection, alcoholic vanillin–sulphuric acid reagent. Sample application (4 µl each): lane 1, eucalyptus oil; lane 2, 1,8-cineole standard; lane 3, lavender oil; lane 4, linalool standard; lane 5, spearmint oil; lane 6, carvone standard; lane 7, 1,8-cineole standard; lane 8, menthol standard

TLC–Direct Bioautography

After TLC separation, the antibacterial activity of the five volatile oils and their main components was detected with direct bioautography. Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. phaseolicola, as plant pathogenic strains, and Staphylococcus epidermidis, S. saprophyticus, S. aureus and methicillin-resistant S. aureus, as human pathogenic strains, were used for bioautography. The minor components of the five essential oils examined in this study had no apparent antibacterial activity at the concentrations at which they were tested. On the whole, the antibacterial activity of essential oils seems to be associated with their most abundant components, but the effect of the minor compounds should also be taken into consideration. In our experiments the main components of the essential oils had only an antibacterial effect in the bioautographic system.

In the oil of thyme, thymol–carvacrol and of the standard, thymol and carvacrol had antibacterial activity (Figure 2) against all the bacterial strains tested. In the oil of cinnamon, trans-cinnamic aldehyde–eugenol and of the standard, eugenol inhibited the growth of all test bacteria. Linalool, in both the oil of lavender and as a standard, showed antibacterial effect only in the case of Staphylococcus saprohyticus. In the TLC-bioautographic system, the essential oil of eucalyptus and spearmint and the standards 1,8-cineole, carvone, menthol and borneol had no antibacterial activity against either plant or human pathogenic bacteria tested in this study (data not shown).

Figure 2.

Detection of the antibacterial activity of thyme and cinnamon essential oils by direct bioautography. Test bacterium, methicillin-resistant Staphylococcus aureus (MRSA). Adsorbent, silica gel 60 F254. Solvent, toluene–ethyl acetate, 93 + 7. Sample application (4 µl each): lane 1, thyme oil; lane 2, thymol standard; lane 3, carvacrol standard; lane 4, linalool standard; lane 5, 1,8-cineole standard; lane 6, borneol standard; lane 7, cinnamon oil; lane 8, eugenol standard

The antibacterial activity of the essential oils and the authentic standards was expressed as the diameter (mm) of inhibition zones (Table 1). All experiments were performed in triplicate.

Table 1. Antibacterial activity, expressed as the diameter (mm) of inhibition zones, of authentic reference of thymol, carvacrol, linalool, eugenol and the main components (thymol-carvacrol, linalool, trans cinnamic aldehyde-eugenol) identified in the essential oil of thyme, lavender and cinnamon, detected by direct bioautography. The values are averages of three parallel measurements
Name of oilThyme oilLavender oilCinnamon oil
Bacterial strainThymol + carvacrolThymol standardCarvacrol standardLinaloolLinalool standardtrans-Cinnamic aldehyde + eugenolEugenol standard
Xanthomonas campestris pv. vesicatoria966No inhibitionNo inhibition66
Pseudomonas syringae pv. phaseolicola866No inhibitionNo inhibition66
Staphylococcus epidermidis877No inhibitionNo inhibition77
Staphylococcus saprophyticus8675577
Staphylococcus aureus (HNCMB 112002)956No inhibitionNo inhibition77
Methicillin-resistant Staphylococcus aureus956No inhibitionNo inhibition87

Among the test bacteria, Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. phaseolicola were the most sensitive. When TLC plates were dipped in an aqueous solution of MTT, the blue background appeared after as little time as 2 h and the spots of the zones of inhibition were clearly apparent, which makes the method faster and more sensitive. Some of the solvents tested (e.g. toluene, ethyl acetate, ethanol, chloroform) applied in TLC separation as mobile phase components had no inhibiting effect in the direct bioautographic system, thus they did not influence the detection of biological activity.

Discussion

Antimicrobial screening methods in essential oil research show great variety.[15] The lack of standardized assays makes direct comparison of results between studies impossible. Therefore there is a need for elaborating standardized, reproducible and reliable methods. We succeeded in optimizing the direct bioautographic method for studying the antibacterial activity of some essential oils. Two plant and four human pathogenic bacteria were used as test microorganisms. The bioautographic method was used for these strains, but our experience suggests that attention should be paid to the different incubation times and temperatures, which was also confirmed in a previous study.[8] This assay is more cost-effective and compares better with traditional microbiological laboratory methods, and is also suitable for analysis of complex, viscous, lipophilic extracts. Interest in the idea of using volatile oils against pathogenic microbes keeps growing, because their side-effects are not significant and often the main effect intended can be achieved. The individual components of the essential oils clearly had antibacterial properties,[16–18] although the mechanism is poorly understood.

There can be no doubt that direct bioautography combined with TLC separation has a novel and expanding field of application and practical advantage in research on antibiotic substances. Bioautography is a valuable technique that can quickly determine which component of an essential oil imparts antibacterial activity. Further investigation need to focus on modelling complex antimicrobial effects in vitro and in vivo.

Acknowledgements

We would like to thank József Németh, head of the Bacteriological Laboratory of Plant Health and Soil Conservation Service of Baranya County, for the plant pathogenic bacterial strains. This work was supported by a grant from PTE ÁOK KA.

Ancillary