Biological activities and compositions of the essential oil of Thymus ciliatus from Algeria

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1 Available online at Scholars Research Library Der Pharmacia Lettre, 2013, 5 (1):28-32 ( ISSN USA CODEN: DPLEB4 Biological activities and compositions of the essential oil of Thymus ciliatus from Algeria Ghorab H 1, Kabouche A 1, Semra Z 1, 2, Ghannadi A 2, Sajjadi EB 2, Touzani R 4 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 Department of Pharmacognosy, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan 81746D73461, Iran 4 LCAE-URAC18, Faculté des Sciences, Université Mohammed Premier, B.P.717, 60000Oujda, Marocco & Université Mohammed Premier Faculté Pluridisciplinaire de Nador, Maroc ABSTRACT The chemical composition of the hydrodistilled essential oil of Thymus ciliatus, collected from Ain Mlila-Province of Oum El Bouaghi (Eastern Algerian), was analyzed by GC and GC/MS. 28 components representing 98.49% of the essential oil were detected with thymol (54.98%), γ-terpinene (11.33%), p-cymene (6.66%) and carvacrol (4.96%) 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 ciliatus, Antioxidant activity, Linoleic acid, β-carotene, Antibacterial activity. INTRODUCTION Essential oils are valuable natural products used as raw materials in many fields, including perfumes, cosmetics, phytotherapy, spices and nutrition [1]. Thymus genus (Lamiaceae) is widely distributed; it comprises eight sections distributed into about 220 species. In North African folk medicine, Thymus plants are used as remedies in various diseases e.g. bronchitis, pulmonary infection, flu, cough and some gastrointestinal disorders [2]. In Algerian flora, there are 12 Thymus species from which 9 are endemic [3]. In continuation of our works on Lamiaceae [4-17], we report here the GC and GC/MS analyses, the antibacterial and the antioxidant activities of the essential oil of the endemic specie Thymus ciliatus collected from Ain Mlila-Province of Oum El Bouaghi (Eastern Algerian). MATERIALS AND METHODS Plant Material Fresh aerial parts of Thymus ciliatus, growing in Ain Mlila (Southern Oum El Bouaghi) was collected in May Voucher specimen wwas deposited at the herbarium of the University of Constantine 1, Algeria (LOST TcAin/05/12). 28

2 Extraction of the essential oil The hydrodistillation of fresh aerial parts (100 g) of Thymus ciliatus, collected from Ain Mlila, for 3h in a Clevenger-type apparatus, according to the British Pharmacopeia, yielded 2 % of a yellow good smell essential oil. Gas chromatography-mass spectrometry (GC/MS) analysis The oil was analyzed by GC/MS using a Hewlett Packard 6890 mass selective detector coupled with a Hewlett Packard 6890 gas chromatograph, equipped with a cross-linked 5% PH ME siloxane HP-5MS capillary column (30 m \ 0.25 mm, film thickness 0.25 µm). Operating conditions were as follows: carrier gas, helium with a flow rate of 2 ml/ min; column temperature, 60D275 C at a rate of 4 C/min; injector temperature, 280 C; injected volume, 0.1 µl of the oil; split ratio, 1:50. The MS operating parameters were as follows: ionization potential, 70 ev; ion source temperature, 200 C; resolution, 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 [18, 19] 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, Proteus mirabilis, Streptococus and. 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) [20]. Antioxidant activity: β-carotene bleaching test The antioxidant activity of the essential oil was evaluated by β-carotene linoleic acid model system [21]. 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, a is the absorbance at time 0, b is the absorbance at time t (120 min) [22]. 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 ciliatus, collected from Ain Mlila, yielded 2% (w/w) of a yellowish good smell oil. 28 compounds were identified by GC and GC/MS, representing 98.49% of the total essential oil mainly, characterized by the main presence of thymol (54.98%) and carvacrol (4.96%). Previously reported essential oils of Algerian species T. ciliatus were thymol chemotype (79.1%, 44.2%) [15, 23] while the essential oil of the species growing in Morocco, were reported to be carvacrol chemotype ( %) [24, 25]. In addition, the present oil was characterized by the main presence of γ-terpinene (11.33%) and p-cymene (6.66%) which were also found to be the main components of the species growing in Eastern Algerian and Morocco ( %) and ( %) [15, 24], respectively. From these results, the present oil seems to be the second highest-thymol content for Thymus ciliatus species, after the essential oil grown at Eastern Algerian [15] which was found to be the highest-thymol content (79.1%) [15]. 29

3 Table 1: Chemical composition, Retention indices and percentage composition of the essential oil of Thymus ciliatus collected from Ain Mlila. Pic Compounds a RI b (%) α-thujene α-pinene camphene sabinene β-myrcene 3-octanol α-phellandrene δ-3-carene α-terpinene p-cymene β-phellandrene 1,8-cineole Trans-β-ocimene γ-terpinene cis-sabinene hydrate linalool α-terpinolene borneol 4-terpineol β fenchyl acetate thymyl methyl ether neral thymol carvacrol β- bourbonene germacrene D caryophyllene oxide α-muurolene 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 exhibited the best antibacterial activity against Enterobacter aerogenes, E. coli ATCC, P. aeruginosa ATCC, Staphylococcus aureus ATCC, E. coli and Staphylococcus aureus with 25 mm, 23 mm, 20 mm, 20 mm, 20 mm, 20 mm inhibition zone diameters respectively (Table 2). Table 2: Antibacterial activity (inhibition zones and MIC) of the essential oil of Thymus ciliatus collected from Ain Mlila. Microrganism Inhibition zone a (mm) MIC (µg/ml) Escherichia coli ATCC Pseudomonas aeruginosa ATCC Staphylococcus aureus ATCC Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus Enterobacter aerogenes Klebsiella pneumoniae Proteus mirabilis Streptococus 17 - 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 [26]. In this study, total antioxidant activity by β-carotene bleaching method [21] assays of the essential oil of Thymus ciliatus, collected from Ain Mlila, was carried out. The activity was increased as dose dependent. The essential oil exhibited a high activity being close effectiveness to the standards. The best inhibition (87.39 %) was measured at 4 mg/ml (Figure 1). 30

4 Figure (1): Inhibition (%) of lipid peroxidation of Thymus ciliatus essential oil and Vitamin E by the β-carotene bleaching method. CONCLUSION The essential oil of Thymus cilatus, collected from Ain Mlila-Province of Oum El-Bouaghi (Eastern Algerian), is mainly characterized by the presence of thymol, p-cymene, carvacrol and γ-terpinene. The present essential oil is thymol chemotype (54.98%) but it s lower than the oil collected from Constantine which is, for the best of our knowledge, the highest-thymol content (79.1%). From these results, the present oil seems to be the second highest-thymol content, for Thymus ciliatus species. Antibacterial assays, showed that the essential oil was more active against Escherichia coli ATCC 25922, Enterobacter aerogenes, P. aeruginosa ATCC, Staphylococcus aureus ATCC, Escherichia coli and Staphylococcus aureus. The essential oil exhibited a high antioxidant activity being close effectiveness to the standards, 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] M. Lahlou, Flav. Fragr. J., 2004, 19, [2] J. Bellakhdar, Pharmacopée marocaine, Ibiss press, France, [3] P. Quezel P, S. Santa, Nouvelle Flore de l Algérie et des Régions Désertiques Méridionales. C.N.R.S., Paris, France, 1963, [4] O. Touafek, A. Nacer, A. Kabouche, Z. Kabouche, C. Bruneau, Chem. Nat. Comp., 2004, 40, [5] A. Ghannadi, E. Sejjadi, A. Kabouche, Z. Kabouche, Z. Naturforsch C., 2004, 59c, [6] A. Kabouche, N. Boutaghane, Z. Kabouche, E. Seguin, F. Tillequin, K. Benlabed, Fitoterapia, 2005, 76, [7] A. Kabouche, Z. Kabouche, C. Bruneau, Flav. Fragr. J., 2005, 20, [8] A. Kabouche, Z. Kabouche, E. Seguin, F. Tillequin, C. Bruneau, Biochem. Syst. Ecol., 2005, 33, [9] A. Kabouche, O. Touafek, A. Nacer, Z. Kabouche, C. Bruneau, J. Essent. Oil. Res., 2006, 18, [10] A. Kabouche, Z. Kabouche, M. Oztürk, U. Kolak, G. Topçu, Food. Chem., 2007, 102, [11] A. Kabouche, Z. Kabouche, S.E. Sajjadi, A. Ghannadi, J. Essent. Oil. Res., 2007, 19, [12] A. Kabouche, Z. Kabouche, Studies in Natural Products Chemistry, Edited by Atta-u-Rahman, Elsevier, 2008, 35, [13] S. Laggoune, A. Kabouche, Z. Kabouche, M. A. El-Azzouny, J. Essent. Oil. Res., 2009, 21, [14] U. Kolak, A. Kabouche, M. Oztürk, Z. Kabouche, G. Topçu, A. Ulubelen, Phytochem. analysis, 2009, 20, [15] A. Kabouche, A. Ghannadi, Z. Kabouche, Nat. Prod. Com., 2009, 4, [16] H. Lakhal, A. Kabouche, Z. Kabouche, Chem. Nat. Comp., 2010, 46,

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