Chemical composition, antimicrobial and antioxidant activities of the essential oils of Œnante virgata Poiret aerial parts, endemic in North Africa

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1 , ISSN Vol. 2, No. 2, pp , June 2012 RESEARCH ARTICLE Chemical composition, antimicrobial and antioxidant activities of the essential oils of Œnante virgata Poiret aerial parts, endemic in North Africa Amar ZELLAGUI 1, Noureddine GHERRAF 1, Salah AKKAL 2 * 1 Laboratory of Biomolecules and Plant Breeding, Life Science and Nature Department,Faculty of Exact Science and Life Science and Nature, University of Larbi Ben Mhidi Oum El Bouaghi,Algeria 2 Laboratoire de Phytochimie et Analyses physicochimiques et Biologiques, Département de Chimie, Faculté de Sciences exactes, Université Mentouri Constantine, Route d Ain el Bey, Constantine *Corresponding author, Tel/Fax: Article History: Received 13 th May 2012, Revised 7 th June 2012, Accepted 8 th June Abstract: Essential components of the aerial parts of Œnante virgata have been studied by gas chromatography-mass spectrometry to afford 27 component, the major components were found to be, Citral (41.71%), α -Bisabolol (21.81%) Dihydrocarviol acetate (11.37%). The antimicrobial activitiy of the essential oils were evaluated by disc diffusion method and tested against Gram-negative Escherichia coli, Staphylococcus aureus and Gram-positive Pseudomonas aerogenosa and Klebsiella pneumonia bacteria. The essential oil showed a strong antibacterial activity against, Escherichia coli, Staphylococcus aureus and Klebsiella pneumonia at low concentration 25 µg/ml. The essential oils were screened for their possible in vitro antioxidant activity by DPPH free radical-scavenging test. The findings showed that percentage of reduction is at 1M. Keywords: Œnante virgata; essential oils; antibacterial activity; antioxidant activity. Introduction Essential oils are important natural products used for their flavour and fragrances in food, pharmaceutical and perfumery industries. They are also sources of aroma chemicals. Biological and pharmacological activities of essential oils and their constituents have been gathering momentum in recent years. Essential oils therefore will continue to be indispensable natural ingredients (Cosge et al. 2009). In the last few years, there has been target interest in biologically active compounds, isolated from plant species for the elimination of pathogenic microorganisms, because of the resistance that microorganisms have built against antibiotics ( Bakkali et al. 2008). Furthermore, many studies point to strong antioxidant activities of aromatic plants and their essential oils. Antioxidant activities are also confirmed for most of the phenolic compounds present in different spices and herbs (Blumenthal et al. 1999; Bruneton 1999). Œnante virgata belongs to umbelifereae family, which is widely spread in North Africa. This plant is abundant in east of Algeria, and no chemical and biological investigations have been reported on this plant. The genus Œnante is represented in Algeria by six species (Quezel and Santa 1963). The main objectives of this study were to determine the chemical composition of its hydro-distilled essential oil by GC/ MS, to investigate the antibacterial activity of the essential oil of Œnante virgata by disc diffusion method against some pathogen strain bacteria and to evaluate the antioxidant capacity of the essential oils. Materials and methods Plant material The aerial parts of Œnante virgata were collected in May 2009 at Mila, AlgeriaThe plant were identified by Prof. M. Kaabache, faculty of Sciences, University of Farhat abbas, Setif Al- *Corresponding author: ( ) salah4dz <A.T.> yahoo.fr 2012 Open Access Science Research Publisher

2 geria, voucher specimens were deposited at the chemistry department University of Mentouri- Constantine under code number ZA 113. Extraction Essential oils were obtained by hydrodistillation of 150g of dried aerial parts using a Clevenger-type apparatus for 3 h. di ethyl ether (10 ml) was used as the collector solvent as reported in literature. After evaporation of the solvent, the oil was dried over anhydrous sodium sulfate and stored in sealed vials protected from the light at 20 C before analyses. Three oil sample were obtained by hydrodistillation and subsequently analyzed by GC-MS. Identification of components Gas chromatography/mass spectroscopy GC/MS analysis was carried out on a Thermoquest-Finnigan Trace GC/MS instrument equipped with a DB-1 fused silica column (60 m, 0.25 mm i.d., film thickness 0.25 m). The oven temperature was raised from 60 to 250 C at a rate of 5 C/min then held at 250 C for 10 min; transfer line temperature was adjusted at 250 C. Helium was used as the carrier gas at a flow rate of 1.1 ml/min with a split ratio of 1/50. Identification of the constituents of each oil was achieved by comparison of their mass spectra and retention times (R t ) with those reported in the literature, and those of authentic samples. Antibacterial activity The Antibacterial activity of the essential oils was evaluated by disc diffusion method using Mueller-Hinton Agar for bacteria with determination of inhibition zones. Microorganism strains All of the bacteria (standard strains; E.coli ATCC 25922, Pseudomonas aerugenosaatcc Staphylococcus aureous ATCC25923 and clinical strains; Klebsella pneumonia were 236 obtained from Bacteriology Laboratory Constantine Hospital University (C.H.U). Antimicrobial assay The Anti-microbial assay was carried out on essential oil extract using agar diffusion method [NCCLS], against four human pathogenic bacteria, including Gram positive (Staphylococcus aureous ATCC25923 and Pseudomonas aerugenosa ATCC 27853), Gram-negative bacteria (Escherichia coli ATCC and klebsella pneumonia). The bacterial strains were first grown on Muller Hinton medium (MHI) at 37 C for 24 h prior to seeding on to the nutrient agar and the fungal strains at 30 C for 48 h. The essential oil was mounted on sterile filter paper discs (6 mm in diameter) with the following concentrations (µg/ml) 2000, 500, 100, and 25). The discs were placed on the inoculated agar media. The treated Petri discs were kept at 4 C for 1 h, and incubated at 37 C for 24 h. The antibacterial activity was assessed by measuring the zone of growth inhibition surrounding the discs. Each experiment was carried out in triplicate. Antioxidant activity DPPH radical-scavenging activity The capacity of essential oil extracted from O. virgata to reduce the radical 2,2-diphenyl-1- picrylhydrazyl (DPPH) was a ssessed using the method of Masuda et al. (1999). 15 µl of th e essential oil at different concentrations was added to 15 µl L of a DPPH ethanolic solution. The mixture was shaken vigorously and left standing at room temperature for 30 min in the dark. The absorbance of the resulting solution was then measured at 517 nm. The normal purple color of DPPH will turn into yellow when its singlet electron is paired with a hydrogen atom coming from a potential antioxidant. The scavenging activity of essential oil was evaluated according to the formula: DPPH scavenging effect (%) = [(A 0 A 1 )/A0] 100

3 Where A 0 is the absorbance of the control at 30 min, and A1 is the absorbance of the sample at 30 min. All samples were analyzed in three replications. Results and discussion GC-MS analysis The composition and percentage of the compounds are summarized in Table 1. They are listed by their order of retention times. The oil yield was 1.20 % (w/ w) based on the dried weight which means that the organs are a potential oil source. Twenty six compounds were identified in the essential oils, representing % of the total oil. The essential oils were dominated by a large amount of oxygenated Monoterpenes (44.48 %) and oxygenated Sesquiterpenes (22,11 %), while the Hydroca r- bons Sesquiterpenes 1.94 % and Monoterpene hydrocarbons 0,98 % contents was very low. The main constituents of the essential oil were found to be, Citral (41.71%), α-bisabolol (21.81%), Dihydrocarviol acetate (11.37%) and some other compounds were only present in minor amounts. In total, essential oil composition of Œnante virgata was considered as a rich source of oxygenated Monoterpenes and oxygenated Sesquiterpenes. Concerning the chemical composition of the essential oil of other Œnante species, (Lansheng et al.(2009) showed that the oils of Œnante javanica (BL.) DC were dominated by allyl phenoxyacetate (80.17%), eudesma -4(14),11- diene (6.83%), 2,3 -dihydro-3-methyl-3- benzofuran-methanol (2.94%), limonene (1.63%). In the aerial parts of Œ. crocata L., Bonsignore et al.(2004) also found α- terpinolene (28.1%), dl -limonene (16.0%), γ- terpinene (10.3%), β-pinene (9.7%) and α- pinene (6.0%) as the main constituents.in add i- tion Pino et al. (2004) reported that the essential oil of Œ. divaricata (R. Br.) Mabb characterized by high contents of β-bisabolene (17.7%), limonene (16.0%) and β-caryophyllene (15.3%) were the major components. Similar results have been found by our studies from Œ. virgata, but very different from that of all the other Œnante species studied so far. 237 Table 1: Chemical composition of volatile oil from Œnante virgata. Compounds Rt % limonene ,9-tetradecadiyne Isomercenol α- farnesene ocimene α-methyl pentenal ,3,6,octatriene,3,7-dimethyl linalol Nerolidol Myrcenol α-bisabolol Dihydrocarveol acetate Megastigma,3,7(z),9,triene ,6octadecadenoicacid,methyl ester Xanth or α-farnesene citral Xanth Acetic acid cyano ,octin-2-ol Acetic acid, cyano octadecanal Venyl hexyl ether Table 2: Classification of the constituents of the Œnante virgata No Component Esters Aliphatic hydrocarbons cyclic hydrocarbons ethers alcohols oxygenated Monoterpenes Monoterpene hydrocarbons Aldehydes oxygenated Sesquiterpenes Hydrocarbons Sesquiterpenes Nitrogen containing compounds Others Antibacterial activity Peak area A wide variety of essential oils are known to possess antimicrobial properties and in many cases this activity is due to the presence of active constituents, mainly attributable to isoprenes such as monoterpenes, sesquiterpenes and related alcohols, other hydrocarbons and phenols The lipophilic character of their hydrocarbon skeleton and the hydrophilic character of their functional groups are of main importance in the antimicrobial action of essential oil components (Griffin et al. 1999), while in other cas-

4 es the Essential oils rich in phenolic compounds are reported to possess high level of antimicrobial activity (Sivropoulou et al.1995; Tepe et al. 2005). Due to these data we were interested to study antibacterial activity of the essential oils. The results summarized were in Table 2 which showed that essential oil extracted from Œnante virgata prevented the growth of all the tested microorganisms with an inhibition zone medium diameter increasing proportionally with the concentrations of the tested samples. The obtained inhibition on bacteria strains varied from 6.5 to 24 mm with a highest inhibition zone recorded 238 with E. coli ATCC25922 at µg/ml, and a considerable inhibition effect with the same concentration at Klebsiella pneumonia and Staphylococcus aureous ATCC25923 It should be mentioned that there are no background antibacterial study on Œnante virgata While in genus Œnante some studies have been reported. The oil of Œnante crocata L. showed a moderate antibacterial activity against Streptococcus faecalis and Bacillus lentus ( Bonsignore et al. 2004). Also the essential oil of O. javanica exhibited in vitro antibacterial activity against some pathogen bacteria (Sharma et al. 1988). Table 3: Inhibition Effect of Essential oils from Œnante virgata. 250µg/ml 500µg/ml 1000µg/ml 2000µg/ml 4000µg/ml 8000µg/ml E. coli ATCC ± ± ± ± ± ±1.25 Pseudomonas aerogenosaatcc ±0.57 8±1.70 9±1.73 9± ± ±1.41 Staphylococcus aureous ATCC ± ± ±0.5 12± ± ±0.81 Klebsiella pneumonia ± ± ± ± ± ±0.57 Antioxidant activity The antioxidant activity of essential oils is another biological property of great interest because they may preserve foods from the toxic effects of oxidants. Moreover, essential oils being also able of scavenging free radicals may play an important role in some disease prevention such as brain dysfunction, cancer, heart disease and immune system decline. (Miguel 2010). Free radical scavenging by using DPPH, essential oil for DPPH radical scavenging activity at a concentration of 10-1 M,10-2 M,10-3 M,10-4 M. Vitamin C was used as standard at the final concentration of 1 mg/ml. The highest DPPH radical scavenging activity (%) was shown by essential oil at 1M (13.22 %) which exerted lowest antioxidant activity than standard vitamin C (71%), fig.1. the chemistry of separate plant organs. The occurrence of citral and α-bisabolol in the essential oil of Œnante virgata and the low extensiveness of a similar volatiles production in genus Œnante represents one of the most important chemical features of Œnante virgata and clearly characterize it from the other Œnante species. An additional characteristic of Œnante virgata oil was its prominent antibacterial activity against some pathogenic microorganisms. Conclusions Our results demonstrate the high chemical variability of constituents found in Œnante virgata and genus Œnante in general and stresses the importance of investigations dealing with Figure 1: Scavenging activity of essential oils against the DPPH radical at different concentrations.

5 Figure 2: Scavenging activity of essential oil and vitamin C against the DPPH radical at different concentrations Acknowledgements : Authors are grateful for the partial financial support by The MESRS (Ministère de l Enseignement Supérieur et de la Recherche Scientifique) Algeria. References Bakkali, S., Averbeck, D., Averbeck, M Biological effects of essential oils A Review, Food and Chemical Toxicology. 46: Blumenthal, M The Complete German Commission E Monographs; American Botanical Council: Austin, TX, USA, Bonsignore, L., Casu, L., Loy, G., Deidda, D., Genco, F Analysis of the essential oil of Oenanthe crocata L. and its biological activity. Journal of Essential Oil Research, 16: Bruneton, J Pharmacognosy, Phytochemistry, Medicinal Plants, 2nd ed.; Intercept Ltd.: London, UK, Cosge, B., Turker, A., Ipek A., Gurbuz B. and Arslan N Chemical Compositions 239 and Antibacterial Activities of the Essential Oils from Aerial Parts and Corollas of Origanum acutidens (Hand.-Mazz.) Ietswaart, an Endemic Species to Turkey Molecules, 14: Cosge, B., Turker, A., Ipek, A., Gurbuz, B., Arslan, N Chemical Compositions and Antibacterial Activities of the Essential Oils from Aerial Parts and Corollas of Origanum acutidens (Hand.-Mazz.) Ietswaart, an Endemic Species to Turkey. Molecules, 14: Griffin, S.G., Wyllie, S.G., Markham, J.L., Leach, D.N Flavour Fragrance J. 14:322. Masuda, T., Yonemori, S., Oyama, Y., Takeda, Y., Tanaka, T., Andoh, T Evaluation of the antioxidant activity of environmental plants: Activity of the leaf extracts from seashore plants. Journal of Agriculture and Food Chemistry, 47: Miguel, M. G Antioxidant and Anti- Inflammatory Activities of Essential Oils: A Short Review. Molecules, 15: Quezel, P. Santa, S Nouvelle flore d Algérie et des régions désertiques méridionales. Paris, CNRS, Sharma, S. K., Singh, V. P., Bhagwat, R In vitro antibacterial effect of the essential oil of Oenanthe javanica (Blume) DC. Indian Journal of Medical Research, 71: Sivropoulou, A., Kokkini, S., Lanaras, T Journal of Agricultural and Food Chemistry, 43: Tepe, B., Daferera, D., Sokmen, A., Sokmen, M., Polissiou, M Food Chem. 90: