Composition, antibacterial and antioxidant activity of the essential oil of Thymus numidicus Poiret from Constantine (Algeria)

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1 Available online at Scholars Research Library Der Pharmacia Lettre, 2013, 5 (3): ( ISSN USA CODEN: DPLEB4 Composition, antibacterial and antioxidant activity of the essential oil of Thymus numidicus Poiret from Constantine (Algeria) Zeghib A 1, Laggoune S 1, Kabouche A 1, Semra Z 1, 2, Smati F 2, Touzani R 3 and Kabouche Z 1* 1 Université de Constantine 1, Département de chimie, Laboratoire d Obtention de Substances Thérapeutiques (LOST), Constantine, Algeria 2 CHU Benbadis-Constantine, Service de Bacteriologie, Constantine, Algeria 3 LCAE-URAC18, Faculté des Sciences, Université Mohammed Premier, B.P.717, 60000Oujda, Morocco & Université Mohammed Premier Faculté Pluridisciplinaire de Nador, Maroc ABSTRACT The chemical composition of the hydrodistilled essential oil of Thymus numidicus Poiret, collected from Constantine (Eastern Algerian), was analyzed by GC and GC/MS. 33 components representing 98.14% % of the essential oil were detected with thymol (23,92%), linalool (17.20%), o-cymene (11.41%), γ-terpinene (10.84%), thymol methyl ether (6,73%) and carvacrol (6,02%) as the major components. The antibacterial activity of the essential oil was tested against 10 gram-positive and gram-negative bacteria by the use of the disc diffusion method. The antioxidant activity was also investigated by the use of β-carotene bleaching method. Keywords: Thymus numidicus Poiret, Antioxidant activity, Linoleic acid, β-carotene, Antibacterial activity. INTRODUCTION Plants of Thymus genus are widely used in North Africa as expectorant, antitussive, antiseptic, antispasmodic, anthelmintic, antifungal, antibacterial, antimicrobial and antioxidant activities [1]. 12 species are distributed in Algeria, nine of them endemic [2]. In continuation of our works on Lamiaceae [3-27], the present study deals with the analysis and the antibacterial potential of the essential oil of T. numidicus, collected from Constantine (Eastern Algerian). MATERIALS AND METHODS Plant Material Fresh aerial parts of Thymus numidicus Poiret, growing at Constantine (Eastern Algerian) was collected in May Voucher specimen was deposited at the herbarium of the University of Constantine 1, Algeria (LOST Tn/05/12). Extraction of the essential oil The hydrodistillation of fresh flowering aerial parts (100 g) of Thymus numidicus collected from Constantine, for 3h in a Clevenger-type apparatus, according to the British Pharmacopeia, yielded 2 % of a yellow good smell essential oil. Gas chromatography: GC analysis was performed on a Shimadzu GC17A gas chromatograph equipped with a split/splitless injector (250 C) and a flame ionization detector (250 C). Retention times for comparison with authentic compounds were measured using a cross-linked DB5-MS column (40 m 0.18 mm, film thickness 0.18 µm). The oven temperature was programmed as isothermal at 60 C for 5 min, then raised to 275 C at 5 C/min and 206

2 held at this temperature for 5 min. Helium was used as the carrier gas at a rate of 1 ml/min. Relative percentage amounts were calculated from peak area without the use of correction factors. Gas Chromatography-Mass spectrometry: Gas chromatography-mass spectrometry: GC-MS was performed using a Shimadzu QP5050 mass selective detector using a cross-linked DB5-MS column (40 m 0.18mm, film thickness 0.18 µm). The oven temperature was programmed as isothermal at 60 C for 5 min, then raised to 275 C at 5 C/min and held at this temperature for 5 min. Helium was used as the carrier gas at a rate of 1 ml/min. 0.1 µl oil was introduced directly into the source of the MS via a transfer line (280 C) with a split ratio of 1:50 and a linear velocity of 30.0 cm/sec. Ionization was obtained by electron impact (70 ev, source temperature 200 C, resolution 1000). Identification of components Essential oil components were identified based on their retention indices (determined with reference to a homologous series of normal alkanes), and by comparison of their mass spectral fragmentation patterns with those reported in the literature [28, 29] and with authentic compounds. Antibacterial activity The antibacterial activity of the essential oil was tested against a range of microorganisms, namely Escherichia coli ATCC 25922, Escherichia coli, Staphylococcus aureus ATCC 43300, Staphylococcus aureus, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa, Klebsiella pneumonia, Enterobacter aerogenes and Morganella morganii. The reference strains were obtained from the Pasteur Institute (Algiers). The other strains were obtained from the laboratory of bacteriology, Benbadis Hospital, Constantine, using conventional methods (clinical isolation) [30]. Antioxidant activity: β-carotene bleaching test The antioxidant activity of the essential oil was evaluated by β-carotene linoleic acid model system [31]. 0.5 mg of β-carotene in 1 ml of chloroform was added to 25 µl of linoleic acid and 200 mg of Tween 40 emulsifier mixture. After chloroform was evaporated under vacuum, 100 ml of distilled water saturated with oxygen were added by vigorous shaking. Four thousand microliters of this mixture were transferred into deferent test tubes containing different concentrations of the sample. As soon as the emulsion was added to each tube, the zero time absorbance was measured at 470 nm using a spectrophotometer. The emulsion system was incubated for 2 h at 50 C. A blank, devoid of β-carotene, was prepared for background subtraction. Vitamin E was used as standards. The bleaching rate (R) of β-carotene was calculated according to the following equation: R ¼ ln(a=b)=t Where ln is the natural log, is the absorbance at time 0, b is the absorbance at time t (120 min) [29]. The antioxidant activity (AA) was calculated in terms of percent inhibition relative to the control, using following equation: AA = [(R control -R sample )/R control ] 100 RESULTS AND DISCUSSION Chemical composition of the essential oil The hydrodistillation of flowering Thymus numidicus, collected from Constantine, yielded 2% (w/w) of a yellowish good smell oil. 33 compounds were identified by GC and GC/MS, representing 98.14% of the total essential oil mainly, characterized by the main presence thymol (23.92%), linalool (17.20%), o-cymene (11.41%), γ-terpinene (10.84%), thymol methyl ether (6,73%) and carvacrol (6.02%) (Table 1). The present composition is different from the previously reported essential oil T. numidicus collected, ten years ago, from Djebel El-Ouahch (Constantine) which was thymol/carvacrol high-content (68.2/16.9%) [8] and from the species (two populations) collected from Eastern Algerian which was mainly characterized by thymol (59.0%, 68.0%), γ-terpinene (8.5%, 4.2%), p-cymene (5.8%, 4.5%) and carvacrol (3.7%, 4.2%) [32]. Among the principal main components of the present essential oil, thymol ( %, 49%), linalool ( %, 4.6%), γ-terpinene ( %, 8%) and carvacrol ( %, 4%), have been found together as main components of the essential oil of T. vulgaris L., cultivated in Estonia and in other European countries [33], and T. vulgaris from Germany [34], respectively. 207

3 Table 1: Chemical composition, Retention indices and percentage composition of the essential oil of Thymus numidicus collected from Constantine. Pic Compounds a RI b (%) α-thujene α-pinene 1-octen-3ol β-myrcene α-phellandrene cis-4-carene o-cymene D-limonene δ-3-carene 1-nonen-3-ol γ-terpinene 1-octanol trans-sabinene hydrate linalool campholenal trans-pinocarveol trans-verbenone camphor borneol terpine-4-ol α-terpineol myrtenol thymol methyl ether thymol carvacrol Z-caryophyllene alloaromadendrene γ-muurolene γ-cadinene Ledol caryophyllene oxide τ-cadinol Agarospinrol diisobutylphtalate Identified compounds Total a Compounds listed in order of their RI b RI (retention index) measured relative to n-alkanes (C 6-C 24) using HP-5MS column Antibacterial activity : The essential oil of the species T. numidicus exerted inhibitory activity against all tested bacterial strains with 16,0±1,4 to 23,8±2,0 mm inhibition zones (table 2) and inhibited more efficiently the growth of Staphylococcus aureus (ATCC), Staphylococcus aureus (SH), Escherichia coli (ATCC) and Klebsiella pneumoniae (SH) with 23.8±2.0, 22.9±0.9, 20.4±0.5 and 19.6±0.3 mm (inhibition zone diameters, respectively). These results are confirmed by the low MIC s values. This activity may be due to the main components synergic effect. Table 2: Antibacterial activity (inhibition zones and MIC) of the essential oil of Thymus numidicus Poiret Microrganism Inhibition zone a (mm) MIC (µg/ml) Escherichia coli (ATCC) 20.4± Pseudomonas aeruginosa (ATCC) 16.4± Staphylococcus aureus (ATCC) 23.8±2.0 3 Escherichia coli (SH) 18.5± Pseudomonas aeruginosa (SH) 16.0± Staphylococcus aureus (SH) 22.9±0.9 3 Enterobacter airogenes (SH) 16.6± Klebsiella pneumoniae (SH) 19.6± Morganella morganii (SH) 16.4± a : (128 µg/ml) Antioxidant activity Species belonging to the Lamiaceae family exhibit high antioxidant activity, and Thymus species have been investigated as potent natural antioxidants. In this study, total antioxidant activity by β-carotene bleaching method assays of the essential oil of Thymus numidicus, collected from Constantine, was carried out. The activity was increased as dose dependent. The essential oil exhibited a mild activity. The best inhibition (52 %) was measured at 4 mg/ml (Figure 1). 208

4 Figure (1): Inhibition (%) of lipid peroxidation of Thymus numidicus essential oil and Vitamin E by the β-carotene bleaching method. CONCLUSION The essential oil of Thymus numidicus, collected from Constantine (Eastern Algerian) is mainly characterized by the presence of thymol, linalool, o-cymene, γ-terpinene, thymol methyl ether and carvacrol as the major components. From these results, the present oil seems to be quite different from the very earlier reported oil from Constantine and a little different from the Algerian Thymus essentials oils. This may be explained by the climate change through the country and through time. Antibacterial assays, showed that the essential oil was more active against Staphylococcus aureus (ATCC), Staphylococcus aureus (SH), Escherichia coli (ATCC) and Klebsiella pneumoniae (SH). The essential oil exhibited a mild antioxidant activity by the use of β-carotene bleaching method. Acknowledgements The authors are grateful to ANDRS and to the DG-RSDT at the MESRS (Ministery of Scientific Research, Algeria) for the financial support. REFERENCES [1] J. Bellakhdar, Pharmacopée marocaine, Ibis press, France, [2] P. Quezel P, S. Santa, Nouvelle Flore de l Algérie et des Régions Désertiques Méridionales. C.N.R.S., Paris, France, 1963, [3] O. Touafek, A. Nacer, A. Kabouche, Z. Kabouche, C. Bruneau, Chem. Nat. Comp., 2004, 40, [4] A. Ghannadi, E. Sejjadi, A. Kabouche, Z. Kabouche, Z. Naturforsch C., 2004, 59c, [5] A. Kabouche, Z. Kabouche, E. Seguin, F. Tillequin, C. Bruneau, Chem. Nat. Comp., 2004, 40 (2), [6] Z. Kabouche, N. Boutaghane, S. Laggoune, A. Kabouche, Z. Ait-Kaki, K. Benlabed, I. J. Aromather., 2005, 15, [7] A. Kabouche, N. Boutaghane, Z. Kabouche, E. Seguin, F. Tillequin, K. Benlabed, Fitoterapia, 2005, 76, [8] A. Kabouche, Z. Kabouche, C. Bruneau, Flav. Fragr. J., 2005, 20, [9] A. Kabouche, Z. Kabouche, E. Seguin, F. Tillequin, C. Bruneau, Biochem. Syst. Ecol., 2005, 33, [10] A. Kabouche, O. Touafek, A. Nacer, Z. Kabouche, C. Bruneau, J. Essent. Oil. Res., 2006, 18, [11] A. Kabouche, Z. Kabouche, S.E. Sajjadi, A. Ghannadi, J. Essent. Oil. Res., 2007, 19, [12] A. Kabouche, Z. Kabouche, M. Ozturk, U. Kolak, G. Topçu, Food Chem., 2007, 102, [13] A. Kabouche, Z. Kabouche, Bioactive diterpenoids of Salvia species. Studies in Natural Products Chemistry, 2008, 35C, [14] S. Laggoune, N. Boutaghane, A. Kabouche, Z. Kabouche, Z. Ait-Kaki, B. Ait-Kaki. Chem. Nat. Comp., 2008, 44(3),

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