African Journal of Microbiology Research Vol. 6(7), pp. 1504-1508, 23 February, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR11.1362 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Chemical composition and in vitro antibacterial activity of clinopodioides Lam. essential oil against some pathogenic bacteria

Soltani Nejad Shahla

Department of Biology, Islamic Azad University, Jiroft branch, Jiroft, .

Accepted 29 December, 2011

The chemical composition of the essential oil obtained from the aerial parts of Ziziphora clinopodioides Lam . was analyzed by gas chromatography-mass spectrometry (GC-MS) method. Twenty six components representing 97.6% of the total oil were identified. The major constituents were pulegone (34.4%), piperitenone (15.1%), 1-8- cineole (6.5%), neo-menthol (5.7%), menth-2-en-1- ol (5.3%), menthol (5.2%), carvacrol (5.1%) and menthone (4.5%). Antibacterial activity of the oil was tested against some human pathogenic bacteria. This oil showed good activity against all tested bacteria, except for Pseudomonas aeruginosa , which is possibly due to the high levels of pulegone in their compositions. However, the essential oil of Z. clinopodioides Lam. is a suitable drug against some human pathogenic bacteria.

Key words: Ziziphora clinopodioides , antibacterial activity, essential oil composition, minimum inhibitory concentration (MIC).

INTRODUCTION that is widespread all over Iran (Mozaffarian, 1996; Salehi There is a popular and scientific interest to screen et al., 2005). Z. clinopodioides Lam. with the common essential oils of used medicinally all over the world Persian name, “Kakuti-e-kuhi” is an endemic species, (Salehi et al., 2005). The use of medicinal plants as grows wild in Iran and also Afghanistan, and Tallish antibacterial and anti-inflammatory drugs in folk medicine (Zargari, 1995). Z. clinopodioides Lam. is an edible is a practice common in Iran, although in most cases, the medicinal plant that its leaves, flowers and stems are active principles of the plants are unknown (Hajhashemi frequently used as wild vegetable or additive in foods to et al., 2002). In recent years, multiple drug resistance in offer aroma and flavor (Zargari, 1995). In Iranian and human pathogenic microorganisms has been developed Turkish folk medicine, Ziziphora species have been used due to the indiscriminate use of commercial antimicrobial as infusion for various purposes such as sedative, drugs commonly used in the treatment of infectious stomachache and carminative (Ozturk and Ercisli, 2007). diseases (Ahanjan et al., 2011). This situation forced In Iranian folklore, the dried aerial parts of this plant have scientists to search for new antimicrobial substances been frequently used as culinary and also in cold and from various sources like medicinal plants (Clark, 1996; cough treatments (Zargari, 1995). Ziziphora spp. has also Cordell, 2000). been used to treat various ailments, such as antiseptic The genus Ziziphora L. belonging to the and wound healing (Ozturk et al., 1995). The family consists of four species ( Ziziphora clinopodioides, composition, antibacterial and antioxidant activity of the , Ziziphora persica and Ziziphora tenuior ) essential oil and various extracts of Z. clinopodioides were reported (Mozaffarian, 1996; Salehi et al., 2005). The purpose of this study was to investigate the chemical composition and antibacterial activity of the essential oil *Corresponding author. E-mail: [email protected]. of Z. clinopodioides against some human pathogenic Tel: +98-913-3471859. bacteria. Shahla 1505

MATERIALS AND METHODS Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) Plant The inocula of the bacteria were prepared from 12 h broth cultures The aerial parts of Z. clinopodioides were collected locally during its and suspensions were adjusted to 0.5 McFarland standard turbidity. flowering stage in June, 2011 from Baft City (Kerman Province, The MIC of Z. clinopodioides essential oil against bacterial strains Iran) at an altitude of 2500 m and were identified. A voucher was determined based on a micro-well dilution method (Swanson et specimen was deposited in the herbarium of the Department of al., 1992). The 96-well plates were prepared by dispensing into Agricultural Faculty of Islamic Azad University, Jiroft branch. each well 95 µl of Muller-Hinton broth and 5 µl of the inoculum. A 100 µl from Z. clinopodioides essential oil initially prepared at the concentration of 4 µg/ml was added into the first wells. Then, 100 µl Essential oil distillation from their serial dilutions was transferred into six consecutive wells. The last well containing 195 µl of Muller-Hinton broth without oil and The dried aerial parts of plant material (100 g) were hydrodistilled 5 µl of the inoculum on each strip was used as negative control. for 3 h using a Clevenger type apparatus. The essential oil was The final volume in each well was 200 µl. Contents of each well collected over water, separated and dried over anhydrous sodium were mixed on a plate shaker at 300 rpm for 20 s and then sulphate, and then was stored in sealed vials at 4°C until chemical incubated in 37°C for 24 h. Microbial growth was det ermined by analysis and antimicrobial screening. The essential oil isolated was absorbance at 600 nm. The lowest concentration inhibiting growth obtained in yield of 0.96% (w/w) based on dried material. was regarded as the MIC of the essential oil (Sahin et al., 2003). The MIC for each of the test bacteria was determined in triplicate assays. In order to confirm MICs and to establish MBCs, 10 µl of Bacterial strains broth was taken from each well and inoculated on Muller-Hinton agar (MHA) plates. The agar plates were incubated at 37°C for 24 h The microorganisms used in this study were three Gram positive, and observed for absence or presence of growth. The minimum Staphylococcus aureus PTCC 1431, Listeria monocytogenes PTCC concentration preventing visible growth of the organisms was taken 1298, Bacillus cereus PTCC 1015 and three Gram negative, as MBC (Cosentino et al., 1999). All procedure was performed in Pseudomonas aeruginosa PTCC 1430, Salmonella enterica PTCC triplicates. 1709, Enterobacter aerogenes PTCC 1221, provided from the Institute Pasture of Iran. RESULTS AND DISCUSSION

Gas chromatography-mass spectrometry (GC-MS) analysis Essential oil analysis GC-MS analysis of the oil was conducted using a Hewlett Packard 5890 II gas chromatography equipped with a Hewlett Packard 5971 The results obtained in the qualitative and quantitative mass selective detector. Analytic conditions; injector and transfer analyses of essential oil are shown in Table 1. Twenty six line temperatures, 220 and 240°C, respectively; oven temperature programmed from 70 to 240°C at 4°C/min; carrier gas, h elium at 1 compounds were identified in the oil of Z. clinopodioides , ml/min; injection of 0.2 ml (10% hexane solution); split ratio, 1:30. representing 97.6% of the total oil. The main constituents The ionization of the sample components was performed in the of the essential oil were pulegone (34.4%), piperitenone electron impact (EI) mode at 70 eV of electron energy. (15.1%), 1-8- cineole (6.5%), neo-menthol (5.8%), menth- 2-en-1-ol (5.3%), menthol (5.2%), carvacrol (5.1%), and menthone (4.5%). Identification of components

The linear retention indices for all the compounds were determined by coinjection of the sample with a solution containing the Antibacterial activity of the essential oil homologous series of C8 to C22 n-alkanes (Van Den Dool and Kartz, 1963). The individual constituents were identified by their The results regarding the antibacterial activity of the identical retention indices, referring to known compounds from the essential oil from Z. clinopodioides are indicated in Table literature (Adams, 1995) and also by comparing their mass spectra with either the known compounds or with the Wiley mass spectral 2. The essential oil of Z. clinopodioides showed good database. activity against all test bacteria, except for P. aeruginosa , as compared to ampicilin antibiotic (as positive control).

Antibacterial activity determination Determination of MIC and MBC values The antibacterial activity of the essential oil of Z. clinopodioides was carried out by disc diffusion method using 100 µl of suspension containing 10 8 CFU/ml of bacteria spread on Muller-Hinton agar The MIC value of the essential oil of Z. clinopodioides for (MHA) medium (Kim et al., 1995). Sterile 6 mm diameter filter paper all test bacteria is as shown in Table 3. The oil inhibited discs were impregnated with 10 µl of essential oil and were placed the growth of E. aerogenes (MIC = 0.25 µg/ml), S. on to Muller-Hinton agar. After 24 h of incubation at 37°C, the enterica (MIC = 0.25 µg/ml), S. aureus (MIC = 0. 5 diameter of the clear zone around the disc was measured and expressed in millimeters as its antibacterial activity. A standard µg/ml), L. monocytogenes (MIC = 0.125 µg/ml) and B. reference antibiotic (ampicillin 10 µg/disc) was used as positive cereus (MIC = 1 µg/ml). P. aeruginosa exhibited control. Each test was run in triplicate and the data were shown as resistance to all the concentrations of the essential oil mean ± standard deviation (SD). used in the study. The Gram-positive microorganisms 1506 Afr. J. Microbiol. Res.

Table 1. Composition of the essential oil of Z. clinopodioides (GC-MS analysis).

S/N Compound Retention index (RI) Relative percentage 1 β-Pinene 977 0.2 2 1- 8-Cineole 1023 6.5 3 γ-Terpinene 1059 1.3 4 Terpinolene 1075 0.2 5 Linalool 1083 0.4 6 Menth-2-en-1 ol 1131 5.3 7 Menthone 1141 4.5 8 Menthofuran 1145 0.2 9 Neo-menthol 1149 5.8 10 4-Terpineol 1159 0.4 11 Menthol 1172 5.2 12 Isomenthol 1179 1.1 13 Verbenone 1192 2.3 14 Cuminyl acetate 1196 1.8 15 Pulegone 1218 34.4 16 Isomenthone 1222 0.3 17 Piperitone 1226 1.7 18 Carvone 1236 0.8 19 Thymol 1265 2.2 20 Carvacrol 1282 5.1 21 Isomenthyl acetate 1296 0.3 22 Piperitenone 1310 15.1 23 Eugenol 1324 0.4 24 β-Bourbonene 1382 0.2 25 Germacrene 1433 1.2 26 Spathulenol 1561 0.6 Total - - 97.6

Table 2. Antibacterial activity of Z. clinopodioides essential oil.

Inhibition zone Microorganism Essential oil (10 µg/disk) Ampicillin (10 µg/disk) P. aeruginosa na na S. enterica 19.3 ± 0.3 13 ± 0.3 B. cereus 20.3 ± 0.2 14 ± 0.4 E. aerogenes 19 ± 0.5 11 ± 0.3 S. aureus 14 ± 0.5 13 ± 0.3 L. monocytogenes 32.1 ± 0.2 21.3 ± 0.5

na: not active; ±: Standard deviation.

were more sensitive when compared to tested Gram- MBC, indicating a bactericidal action of the oil. negative microorganisms. L. monocytogenes was the most susceptible strain. In the present study, the oil killed 99.9% or more of S. aureus (MBC = 0.5 µg/ml), L. DISCUSSION monocytogenes (MBC = 0.125 µg/ml), S. enterica (MBC = 2 µg/ml) and E. aerogenes (MBC = 2 µg/ml) (Table 4). Plant extracts are extensively used for traditional Although, the MIC and MBC results varied between test medicine in Iran. Essential oils have been used as organisms, in most cases, the MIC was equivalent to the flavoring agents in food and beverages, and due to the Shahla 1507

Table 3. Minimum inhibitory concentration (MIC) of the Z. clinopodioides Lam. oil.

MIC ( µg/ml) Microorganism 0 0.125 0.25 0.5 1 2 4 E. aerogenes + + - - - - - P. aeruginosa + + + + + + + S. enterica + + - - - - - B. cereus + + + + - - - L. monocytogenes + ------S. aureus + + + - - - -

+: bacterial growth. Each test was performed in triplicates.

Table 4. Minimum bactericidal concentration (MBC) of the Z. clinopodioides Lam. oil.

MIC( µg/ml) Microorganism 0 0.125 0.25 0.5 1 2 4 E. aerogenes + + + + + - - P. aeruginosa + + + + + + + S. enterica + + + + + - - B. cereus + + + + + + + L. monocytogenes + ------S. aureus + + + - - - -

+: bacterial growth. Each test was performed in triplicates.

presence of antimicrobial compounds, they have a taurica subsp. cleonioides (81.9%), Z. persica (66%) and potential as natural agents for food preservation Ziziphora vychodceviana (57.5%) (Ozturk et al., 1995). (Helander et al., 1998). Moreover, researchers have been The results of the present study indicated that the interested in biologically active compounds isolated from essential oil of Z. clinopodioides have good antimicrobial plant species for the elimination of pathogenic effect on human pathogenic bacteria. The essential oil microorganisms, because of the resistance they have did not display any antibacterial activity against P. developed to antibiotics (Cordell, 2000). Review of the aeruginosa . It has frequently been reported that Gram (+) published literatures reveal that the composition of Z. bacteria are more susceptible to essential oil than Gram clinopodioides oil from different regions shows large (−) bacteria (Mann et al., 2000). The tolerance of Gram similarity in the major components, but relative (−) bacteria to essential oil has been ascribed to the concentrations have some difference (Aghajani et al., presence of a hydrophilic outer membrane that blocks the 2008; Amiri, 2009; Ozturk and Ercisli, 2007). It is penetration of hydrophobic essential oils into target cell probable that environmental conditions have a significant membrane (Ozturk and Ercisli, 2006). The antibacterial influence on the relative amounts of essential oil activities of the essential oil reported here could be components. The antibacterial activity of the oil may be associated to the presence of (+)-pulegone which was associated with the relatively high pulegone (34.4%), found in about 34.4% in the essential oil. Piperitenone piperitenone (15.1%) and 1- 8-cineole (6.5%) content. It and 1-8- cineole could also be responsible for the activity has been reported that these components have (Baser, 2002; Meral et al., 2002). Previous studies significant antimicrobial activities (Duke et al., 1996). indicate that (+)-pulegone is bactericidal (Marinkovi et al., Also, pulegone is the major component of other species 2002). Pulegone has a similar structure to carvone which of Ziziphora (Ziziphora hispanica, Ziziphora brevicalyx has been shown to affect the cell membrane by and Z. tenuior ) in the published literatures (Meral et al., dissipation of pH gradient and membrane potential of 2002; Sezik et al., 1991). The results of this research are cells (Burt, 2004). in accordance with earlier studies on Ziziphora spp. that were all found to be rich in pulegone (Dembistikii et al., 1995; Meral et al., 2002; Bakkali and Averbeck, 2008). Conclusion Compared to the other Ziziphora spp., pulegone content of the essential oil of Z. clinopodioides Lam. (34.4%) was The results of this study are in agreement to a certain lower than those of Z. tenuior (87.1%), Ziziphora degree with the traditional uses of Z. clinopodioides that 1508 Afr. J. Microbiol. Res.

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