Chemical Composition and Antibacterial Activity of the Essential Oil of Thymus ciliatus (Desf.) Benth. ssp. eu-ciliatus Maire from Algeria

Abstract

Thymus ciliatus ssp. eu-ciliatus harvested in western Algeria produces an essential oil whose composition is dominated by carvacrol (72.4-80.3%). The oil displayed antimicrobial activity.

Key Word Index

Thymus ciliatus ssp. eu-ciliatus, essential oil, ^sup 13^C-NMR, carvacrol, antibacterial activity, antifungal activity, Algeria.

Introduction

All species of Thymus genus (Labiatae famdy) produce an essential oil, and several representatives are important herbs and spices used in ad part of the world. To date, the ods of 162 taxa of the Thymus genus have been chemicaUy investigated revealing about 360 volatiles components in total and wide essential oil polymorphism. The phenolic terpenes, thymol and carvacrol, rank highest in importance (1).

Several species of thyme genus grow wdd in Algeria, among which Thymus ciliatus (Desf.) Benth., which is an endemic species from North Africa. This species includes tdiree subspecies: ssp. eu-ciliatus Maire, ssp. coloratus (Boiss. et Reut.) Batt. and ssp. munbyanus (Boiss. et Reut.) Batt. Thymus ciliatus ssp. eu-ciliatus is widespread in brushwood and lawn, shows littie green leafs, with cdium outside. Flowers are purplish or red, more than 1 cm lengtìi (2). This aromatic herb is used in folk medicine for its anti-spasmodic, diaphoretic and stimulant effects (3).

The composition of 14 oil samples of Thymus ciliatus of Moroccan origin (subspecies not specified) has been reported (4,5). The content of die major components varied substantially from sample to sample: tiiymol (0.3-29.3%), carvacrol (0.4-21.7%), α-terpinyl acetate (0-42.9%), geranyl acetate (0-21.7%), geranyl butyrate (0-26.7%), camphor (0.4-28.4%) and borneol (0.1-31.6%).

On continuation of our research on essential odbearing plants from Algeria (6,7), we investigated the chemical composition and the antimicrobial activity die od from aerial parts oiThymus ciliatus ssp. eu-ciliatus growing wdd in Tlemcen province.

Experimental

Plant Material: Arial parts of Thymus ciliatus ssp. euciliatus were collected in full blossom during May-June 2003 in Imama (sample T1), Aïn el Hadjar (T2), Koudia (T3) and Mansoura (T4) localities and during May-June 2005 in Hammam Schiguer(T5), Sebâa Chioukh(T6), Sebdou (T7) and Remchi (T8) localities (Tlemcen province).

Isolation ofessential oils andfractionation: Aerial parts of 3-4 plants harvested in a restricted area were air-dried and water-distillated widi a Clevenger-type apparatus for 3 h. One sample of oil (Tl, 2 g) was chromatographed over SiO^sub 2^. Four fractions were eluted widi pentane F 1 (302 mg), pentane/diethyl ether (75/25), F2 (662 mg) and diethyl ether, F3 (904 mg) and F4 (120 mg), respectively.

Analytical GC: GC analysis was carried out using a Perkin-Elmer Autosystem apparatus equipped widi FID and two fused-silica capdlary columns (50 m x 0.22 mm i.d., film thickness 0.25 pm), BP-I (polydimethyl sdoxane) and BP-20 (polyetliylene glycol) . The oven temperature was programmed from 60°-220°C at 2°C/min and then held isothermal at 220°C for 20 min; injector temperature: 250°C; detectortemperature: 250°C; carrier gas: He (0.8 mL/min); split: 1/60. The relative proportions of the essential od constituents were expressed as percentages obtained by peak area normalization, without using correcting factors. Retention indices (RI) were determined relative to the retention times of a series of n-alkanes widilinearinterpolation(« Target Compounds » software from Perkin Elmer).

^sup 13^C- NMR Analysis: NMR spectra were recorded i) on a Bruker AC 200 Fourier transform spectrometer operating at 50.323 MHz for ^sup 13^C, equipped widi a 10 mm probe, in CDCl^sub 3^, with all shifts referred to internal TMS. ^sup 13^C-NMR spectra were recorded with the following parameters: pulse widtii (PW), 5 ps (flip angle 45°); acquisition time, 1.3 s for 32 K data table with a spectral width (SW) of 12 500 Hz (250 ppm); CPD mode decoupling; digital resolution 0.763 Hz/pt. In a typical procedure, 200 mg of the oil was dduted in 2 mL of CDCl^sub 3^. The number of accumulated scans ranged between 3,000 and 5,000 for each sample. Exponential line broadening multiplication (LB = 1 Hz) of the free induction decay (FID) was applied before Fourier transformation; or ii) on a Bruker AVANCE 400 Fourier Transform spectrometer operating at 100.13 MHz for ^sup 13^C-NMR, equipped with a 5 mm probe, in CDCl3, with all shifts referred to internal TMS. ^sup 13^C-NMR spectra were recorded with the following parameters: pulse widdi = 4 ps (flip angle 45°); acquisition time = 2.7 s for 128K data table with a spectral width of 25 000 Hz (250 ppm); CPD mode decoupling; digital resolution = 0.183 Hz/pt. The number of accumulated scans was 2000 or 3000 for each sample, essential oil or chromatographic fraction thereof (around 40 mg of the mixture in 0.5 mL of CDCl^sub 3^).

Identification of Components: Identification of the individual components was based: (i) on comparison of tiieir GC retention indices (RI) on polar and apolar columns, determined relative to the retention times of a series of n-alkanes widi linear interpolation widi those of reference compounds (ii) by ^sup 13^C-NMR spectroscopy, fodowing the methodology developed and computerized in our laboratories, using homemade software, by comparison widi spectral data of reference compounds compded in a laboratory-budt library (8). AU the components of sample Tl were identified by ^sup 13^C-NMR and GC(RI) in the fractions of chromatography as wed as the major compounds of the otiier samples. Minor compounds of samples T2-T8 were identified by comparison of their retention indices widi those of the components of sample Tl.