Composition and Antimicrobial Activity of the Essential Oil of Thymus fontanesii.

Abstract

The yield of oil isolated by hydrodistillation from aerial parts of Thymus fontanesii. Boiss. et Reut. (Lamiaceae) growing wild in Djelfa (Algeria) was 0.9%. Analysis of the oil by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) revealed the identification of 47 components accounting for 98.5% of the total oil. The oil was found to be rich in monoterpenes (91.5%) with major constituents being thymol (29.3%), γ.-terpinene (21.7%), p.-cymene (15.9%), and thymol methyl ether (11.4%), while a smaller amount of linalool (4.8%) and β.-caryophyllene (2.9%) were detected. According to the antimicrobial study using the disk diffusion method and the agar dilution assay–minimal inhibitory concentration (MIC), the oil showed strong in vitro. growth inhibition activity against Gram-negative bacteria and antifungal activity. The oil exhibited the maximum antifungal activity against Mucor ramaniamus. (MIC = 0.2 µL/mL).

Keywords:

• Antimicrobial activity
• essential oil composition
• Lamiaceae
• MIC
• p.-cymene
• γ.-terpinene
• thymol
• thymol methyl ether
• Thymus fontanesii

Introduction

Many species of Thymus. (Lamiaceae) have been widely used in folk medicine throughout the world (Bruneton, 1999). It is well-known that Thymus. species are rich in essential oils and are characterized by a remarkable variability of both morphology and oil chemical composition (Adzet et al., 1977; Stahl-Biskup, 1991; Salgueiro et al., 1997). Several recent studies on the biological and microbiological activity of Thymus. oils and methanol extracts have been reported (Mouhajir et al., 2001; Dorman & Deans, 2004; Giordani et al., 2004; Rasooli & Abyanch, 2004; Ozturk & Ercisli, 2005).

The genus Thymus. is represented in Algeria by 12 species. Thymus fontanesii. Boiss et Reut is an odoriferous endemic species in the north of Algeria and Tunisia that grows mostly on lawn soil. In Algeria it is known as “zaateur” and it is an annual plant with dressed robust stems, oblong-lanceolate leaves, and white or pale flowers (Quezel & Santa, 1963). The current work reports the results of detailed chemical composition analysis, antibacterial and antifungal activities of the essential oil of T. fontanesii. collected from the Djelfa region (Algeria).

Materials and Methods

Plant material

The aerial parts (stems + leaves) of T. fontanesii. growing wild were harvested in May 2003 from Djelfa city, Algeria (300 Km south of Algiers; longitude 2°45′; latitude 34°30′; elevation 1143 m; annual precipitation 308 mm; semiarid climate type). Plant identification was carried out by Mr A. Beloued, botanist, Agronomic National Institute, Algiers, Algeria, and voucher specimens have been deposited (HINA/FA/no. P 152).

Extraction and isolation of the essential oil

The shade-dried and finely powdered aerial parts of the plant were exhaustively extracted by hydrodistillation for 3 h using a Clevenger-type apparatus with a water-cooled receiver in order to reduce hydrodistillation overheating artifacts. The oil was extracted from the distillate with diethyl ether and then dried over anhydrous sodium sulfate. After filtration, the solvent was removed by distillation under reduced pressure in a rotary evaporator. Oil was obtained in a yield of 0.9% based on dried weight of sample. The oil was stored in a sealed glass vial in the dark at 4°C until analysis and bioassays tests.

Gas chromatography

Gas chromatography (GC) (J&W Scientific) analysis was performed on a Trace CE Thermo-Finnigan chromatograph using fused silica capillary column with stationary phase DB-5. The various parameters fixed for DB-5 column are: 30 m × 0.32 mm, 0.25-µm film thickness. Oven temperature was programmed from 60°C for 3 min then 3°C/min to 240°C for 5 min. The detector and injector temperatures were 250°C and 260°C, respectively. Nitrogen was used as carrier gas at a flow rate of 1 mL/min in the split mode 1:50, with an injection vol. 0.2 µL. The percentage of composition of the identified components was computed from the GC peak area on DB-5 without any correction factor and was calculated relatively.

Gas chromatography/mass spectrometry

Gas chromatography/mass spectrometry (GC/MS) analysis was performed on a Trace MS Thermo-Finnigan chromatograph apparatus equipped with a DB-5 column (30 m × 0.32 mm, with 0.25-µm film thickness) with helium as carrier gas at a flow rate of 1 mL/min. The GC oven temperature was kept at 60°C for 3 min and programmed to 240°C for 3 min at a rate of 3°C. Injector temperature was 250°C. The mass spectrometer was operating in the EI-mode at 70 eV. The less source temperature was 200°C; acquisition mass range, m/z. 40–450.

Identification of components

The retention indices (RIS) were calculated by comparing the retention times of the eluting peaks with those of the n.-alkanes (C₅–C₂₈) using a Van den Dool and Kratz formula (1963) on DB-5 column. The identification of individual components was based on comparison of their retention indices along with mass spectra to those stored in the spectrometric electronic library (NIST) and with those reported in the literature (Adams, 1995).

Tested microorganisms

For the bioassays, the following collection of eight microbes were used: two Gram-positive (Bacillus subtilis. ATCC 6633, Staphylococcus aureus. CIP 7625) and two Gram-negative (Escherichia coli. CIP 54.8, Pseudomonas aeruginosa. CIP A22) bacteria, two yeasts (Saccharomyces cerevisiae., Candida albicans.), and two filamentous fungi (Mucor ramanianus. NRRL 6606, Fusarium oxysporum. f. sp. albedinis.). All microorganisms were obtained from the Microbiological Laboratory, Department of Biology, Ecole Normale Superieure, Algiers, Algeria. Cultures of the microorganisms were maintained on nutrient agar (NA) medium.

Screening for antimicrobial activity

Diffusion method using filter paper disk was used for the screening of oil antibacterial and antifungal activities. For this, organism suspension was prepared with sterile physiological water uniformly mixed with sterile liquid nutrient agar in Petri dishes. Sterile filter paper disks (9 mm) were soaked with 30 µL of oil and placed on the surface of NA medium. Petri dishes were placed at 4°C for 24 h to allow the diffusion of the oil from disk to medium. After the incubation period (28–30°C for 24 h for bacteria and 28°C for 36–48 h for fungi), the inhibition halo diameters were measured using a ruler and expressed in millimeters.

Determination of the minimum inhibitory concentrations (MICs)

MIC determination of oil was carried out by the agar dilution method. For this, 2 µL (three replicates of each organism) of microbe suspension was placed as drop on the surface of NA medium homogenously containing oil. The different concentrations used in this assay were 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 7.0, 10.0, 12.0, 15.0 µL oil/mLNA. The control received the same quantity of microorganisms without oil. After the incubation period (described above), microbe growth was visually evaluated by comparison with those of control plates. The MIC was taken as the lowest concentration that inhibited growth after incubation.

Results and Discussion

Chemical analyses

The oil was obtained by hydrodistillation of aerial parts of T. fontanesii. with an average yield of 0.9% (w/w) based on dry weight of material. This yield seems to be lower when compared with yield of the oil isolated from the flowering aerial parts (Ghannadi et al., 2004). The different harvesting sites and development phase of plants can partly be responsible for this difference (Bruneton, 1999).

The qualitative and quantitative analytical results obtained by GC and GC/MS are shown in Table 1, where the general chemical profile (compounds are listed in order of their elution on a DB-5 column) of the analyzed oil, the percentage content of the individual components, and retention indices are summarized. Forty-seven compounds, constituting 98.5% of the oil, were identified by their retention indices and mass spectral data. The chemical group distribution of the oil components is reported in Table 2. The oil is mainly composed of oxygen-containing monoterpenes (50.5%), with thymol (29.3%) and thymol methyl ether (11.4%) being predominant. Monoterpene hydrocarbons amount to 41%, with γ.-terpinene (21.7%) and p.-cymene (15.9%) being the major components. The oil contains a lower percentage of sesquiterpene mixture, in which hydrocarbons form 5.6% represented by β.-caryophyllene (2.9%) and very low oxygen containing fraction form 0.7%. The content of some other terpene compounds exceeding 1% in the total oil were linalool (4.8%), borneol (1.9%), α.-terpinene (1.9%), carvacrol (1.8%), and (Z.)-α.-bisabolene (1.5%). The rest of the 37 identified compounds could be detected in the range 0.7–0.1% amounts or less.

Table 1. The chemical constituents of the essential oil of Thymus fontanesii. aerial parts.

Components^a.	RI^b.	GC area (%)
Tricyclene^c.,^d.	918	0.5
α.-Thujene	934	tr^e.
α.-Pinene	939	0.3
Camphene	962	tr
Sabinene^c.,^d.	967	0.3
1-Octen-3-ol	974	0.1
3-Octanone^c.,^d.	979	0.6
Myrcene	985	tr
α.-Phellandrene^c.,^d.	994	0.1
δ.-3-Carene^c.,^d.	1001	tr
α.-Terpinene	1007	1.9
p.-Cymene^f.	1017	15.9
β.-Phellandrene^c.,^d.	1022	tr
1,8-Cineole	1033	tr
γ.-Terpinene	1052	21.7
cis.-Sabinene hydrate	1058	0.3
Terpinolene^c.,^d.	1077	0.1
Linalool	1092	4.8
cis.-Pinene hydrate^c.,^d.	1125	tr
trans.-Sabinol^c.,^d.	1135	tr
Camphor	1139	tr
Borneol	1158	1.9
Terpinen-4-ol^c.,^d.	1169	0.5
p.-Cymene-8-ol^c.,^d.	1177	tr
α.-Terpineol	1188	0.2
Thymol methyl ether	1229	11.4
Carvacrol methyl ether^c.,^d.	1234	0.1
Cymen-7-ol^c.,^d.	1276	tr
Thymol	1290	29.3
Carvacrol	1295	1.8
Eugenol^c.	1343	0.1
α.-Copaene^c.	1360	tr
β.-Caryophyllene^c.,^d.	1404	2.9
α.-Humulene	1438	0.1
allo-Aromadendrene^c.,^d.	1446	tr
γ.-Muurolene^c.	1462	tr
Germacrene D	1467	0.1
Bicyclogermacrene^c.,^d.	1471	tr
α.-Muurolene^c.	1484	0.7
(Z.)-α.-Bisabolene^c.,^d.	1496	1.5
γ.-Cadinene^c.,^d.	1510	0.2
δ.-Cadinene^c.	1524	tr
Spathulenol	1565	0.2
Caryophyllene oxide^c.	1571	0.2
Viridiflorol^c.,^d.	1590	tr
epi-α.-Cadinol^c.,^d.	1637	tr
α.-Cadinol	1641	0.2
Total identified		98.5

GC, gas chromatography.

^a.Compounds listed in order of elution on DB-5 column.

^b.RI (retention index) measured relative to n.-alkanes (C₅–C₂₈) on DB-5 column.

^c.Components reported for the first time in T. fontanesii. essential oil from Algeria.

^d.Components reported for the first time in Algerian Thymus. genus essential oils.

^e.tr, trace (< 0.1%).

^f.Main compounds in bold font.

Table 2. Group composition of Thymus fontanesii. essential oil.

Group of compounds	Content (%)
Monoterpene hydrocarbons	41.0
Oxygen containing monoterpenes	50.5
Sesquiterpene hydrocarbons	5.6
Oxygen containing sesquiterpenes	0.7
Others	0.7
Total identified	98.5

In regard to the one previously reported study of the T. fontanesii. oil (Ghannadi et al., 2004), it is important to point out that there are some important qualitative and quantitative differences. In that oil, 15 compounds representing 99.4% of the total oil were identified and quantified by GC/MS, but in our oil, 47 compounds amounting to 98.5% of the oil were identified by GC (retention indices) and GC/MS but quantified by GC-FID. Twenty-six of these are reported for the first time in Algerian T. fontanesii. oil, and 20 of these components have not been previously reported in other Algerian Thymus. oils (see footnotes c and d in Table 1). However, several studies on North African Thymus. species showed that the main components of the oils were thymol in Algerian T. numidicus. (Kabouche et al., 2005a), thymol in Moroccan Thymus. oils, T. pallidus. and T. zygis. (Richard et al., 1985), and carvacrol from T. capitatus. collected in Tunisia (Hedhili et al., 2002). The above results show that the composition of the essential oil of Algerian T. fontanesii. is quite uniform with those isolated from other Thymus. species, especially the presence of thymol and carvacrol, as well as their biosynthetic precursor p.-cymene and γ.-terpinene. These four components have been previously found as constituents of most Thymus. oils (Stahl-Biskup et al., 1991; Baser et al., 1999; Youdim et al., 2002; Asllani et al., 2003; Barazandeh, 2004). In addition, other components were previously detected as main components and at the same time determine the chemotypes in oils of Thymus. throughout the world, but not detected in our oil, such as geranial (Baser et al., 1995), geraniol and geraniol acetate (Baser et al., 1996; Omidbaigi et al., 2005), α.-terpinyl acetate (Mockute & Bernotiene, 2001), and (E.)-nerolidol (Kulevanova et al., 1998).

Antimicrobial activity

Table 3 shows the antibacterial and antifungal activities (inhibition zone and minimal inhibition concentration) of oil of T. fontanesii. aerial parts against each microorganism tested. This preliminary screening for antimicrobial activity using the paper disk diffusion method showed that the oil exhibited a pronounced antimicrobial effect on some tested organisms. This oil inhibits the growth of all filamentous fungi tested (F. oxysporum. f. sp. albedinis. and M. ramanianus.), all yeast (S. cerevisiae. and C. albicans.), and only one Gram-positive bacterium, B. subtilis., but was unable to inhibit the growth of the Gram-negative bacteria (P. aeruginosa. and E. coli.) and the Gram-positive bacterium S. aureus.. The oil presents the best action as antifungal, in which F. oxysporum. f. sp. albedinis. was the most susceptible fungi (inhibition zone 50 mm).

Table 3. Antimicrobial activity of essential oil of Thymus fontanesii. [inhibition zone and minimal inhibitory concentrations (MICs)].

Microorganisms	Diameter of inhibition zone^a.	MIC (µL/mL)^b.
Gram-positive bacteria
Bacillus subtilis.	44	0.5
Staphylococcus aureus.	–^c.	1.0
Gram-negative bacteria
Escherichia coli.	–	2.0
Pseudomonas aeruginosa.	–	1.0
Filamentous fungi
Fusarium oxysporum. f. sp. albedinis.	50	0.5
Mucor ramanianus.	28	0.2
Yeast
Saccharomyces cerevisiae.	46	1.0
Candida albicans.	32	1.0

^a.Inhibition zone diameter in millimeters around the disk (diameter of the disk, 9 mm, included).

^b.µL of oil/mL of medium.

^c.–, no detectable activity.

The determination of the MIC data (µL of oil/mL of medium) of microbial growth of T. fontanesii. oil against all microorganisms tested by means of the agar dilution method showed that MIC for F. oxysporum. f. sp. albedinis. and B. subtilis. was 0.5 µL/mL, whereas it was very low for M. ramanianus. (0.2 µL/mL). However, MIC of 1 µL/mL was similar for S. cerevisiae., C. albicans., P. aeruginosa. and S. aureus.. E. coli. was the most resistant bacterium (inhibition at 2 µL/mL). It is clear that our oil exhibited an antimicrobial effect against all organism strains tested, where the sensitivity of F. oxysporum. f. sp. albedinis. was also proved. Moreover, we note that P. aeruginosa, E. coli., and S. aureus. have shown a sensitivity to oil, where this sensitivity was not detected by the disk diffusion method. This means that the concentration injected on the paper disk was not sufficient to inhibit the growth of these bacteria. It is obvious that, the dilution method provides much more information than the disk diffusion method.

In both antimicrobial methodologies, T. fontanesii. oil has been found to be effective against bacteria strains and as a strong antifungal, especially against the date palm blight pathogen (fusariose) F. oxysporum. f. sp. albedinis. (Kabouche et al., 2005b). It may be mentioned that this observation was similar to those obtained with T. mastichina. oil against eight Fusarium. species (Fraternale et al., 2003) and with commercial T. vulgaris. oil against other phytopathogenic fungi (Zambonelli et al., 2004). Similarly, many recent reports on essential oils from other Thymus. species have also displayed high levels of antimicrobial activity (Dorman & Deans, 2004; Giordani et al., 2004; Rasooli & Abyanch, 2004).

The antimicrobial activity may, therefore, be due to the presence of strong antimicrobial active components with high percentage in this oil such as thymol, γ.-terpinene, p.-cymene, and linalool (Burt, 2004). In addition, we can advance the assumption on oil activity, already advanced by Zakarya et al. (1993) and Marino et al. (1999), in which minor components produce a synergetic effect among other components. On the other hand, concerning the action mode of oils, Knobloch et al. (1989) suggested that the oil active components, particularly phenolic alcohols or aldehydes, are regarded as biological membrane destructors (interference with the membrane enzymes). Moreover, several other targets in the cell are reviewed recently (Burt, 2004).

Conclusions

In conclusion, it can be observed that the composition of the essential oil from T. fontanesii. growing wild in Djelfa (Algeria) was usual (typical phenolic essential oil: a potential rich source of thymol). The results from our examination and from literature show that oil yield and oil chemical composition, especially the quality and quantity of the main compounds, was very variable. This fact can be attributed to the particular environmental conditions in which the plants were grown, development stage, and the genetic constitution of the plants (Bruneton, 1999).

From the bioassays, we can conclude that T. fontanesii. oil possessed antibacterial activity against Gram-positive as well as Gram-negative bacteria and antifungal activity, although the microorganisms differ in their sensitivity to the oil. These observations lead us to suggest that this oil is a broad spectrum antimicrobial agent.

Acknowledgments

The authors thank Pr. M. Koch and Pr. F. Tillequin, Laboratoire de Pharmacognosie, Faculté de Pharmacie de l'Université René Descartes, Paris, France, for their help and cooperation, and the authors are very grateful to Dr. P. Roland-Gosselin, Thermo-Finnigan, France, for her help and GC/MS technical assistance.