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Journal of Food Processing and Preservation ISSN 1745-4549

ESSENTIAL OIL COMPOSITION AND IN VITRO ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF THYMUS CAPPADOCICUS BOISS. jfpp_694

605..614

1

SEVIL ALBAYRAK and AHMET AKSOY Department of Biology, Science Faculty, Erciyes University, Kayseri 38039, Turkey

1

Corresponding author. TEL: 90-352-4374937/33054; FAX: 90-352-4374933; EMAIL: [email protected] Received for Publication June 13, 2011 Accepted for Publication January 19, 2012 doi:10.1111/j.1745-4549.2012.00694.x

ABSTRACT The present study was conducted to evaluate the phenolic contents, antioxidant and antimicrobial activities of methanol extract (ME), ethanol extract, water extract (WE) and essential oil of Thymus cappadocicus Boiss. The essential oil composition of a hydrodistilled essential oil of T. cappadocicus was analyzed by a gas chromatography/mass spectrometry system. Thymol (70.82%), p-cymene (9.52%), g-terpinene (9.27%) and carvacrol (4.65%) were found to be the main constituents. Several potential antioxidant activities, including phosphomolybdenum, 2,2diphenyl-1-picrylhydrazyl radical scavenging and b-carotene bleaching activity, were evaluated. ME was found to be the most active free radical scavenger. In the b-carotene-linoleic acid system, the extracts and essential oil exhibited strong inhibition against linoleic acid oxidation. The essential oil and extracts were also screened for their antimicrobial activity against 15 microorganisms. The results showed that the essential oil of T. cappadocicus had great antimicrobial activity potential against microorganisms tested. In contrast, the WE showed no antimicrobial activity.

PRACTICAL APPLICATIONS Thymus species are widely consumed as a spice and tea in Turkish traditional medicine. Recently, there has been considerable interest in the extracts and essential oils of aromatic plants with antimicrobial activities for controlling pathogens and/or toxin producing microorganisms in foods. Antioxidants are compounds that, when added to food products, act as radical scavengers, prevent the radical chain reactions of oxidation, delay or inhibit the oxidation process and increase shelf life by retarding the process of lipid peroxidation. In addition, oxidized polyunsaturated fatty acids may induce the progression of a great number of pathological disturbances. Owing to the strong antimicrobial and excellent protective features that they exhibited in antioxidant activity tests, the extracts and essential oil of T. cappadocicus may be considered as a natural source suitable for use in the food and pharmaceutical industries, in addition to its use in alternative medicine and as a natural therapy.

INTRODUCTION Lipid oxidation is a complex free radical chain process involving a variety of radicals and is a major cause of food quality deterioration. Antioxidants have been widely used as food additives to minimize rancidity, retard the formation of toxic oxidation products, maintain nutritional quality and increase

the shelf life of food products (Maisuthisakul et al. 2007). An antioxidant can be defined as any substance that, when present at low concentrations compared with that of an oxidizable substrate, significantly delays or inhibits the oxidation of that substrate (Young and Woodside 2001). However, commonly used synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)

Journal of Food Processing and Preservation 37 (2013) 605–614 © 2012 Wiley Periodicals, Inc.

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BIOACTIVITY OF THYMUS CAPPADOCICUS

S. ALBAYRAK and A. AKSOY

are restricted by legislative rules because they are suspected of having some toxic effects and of being possible carcinogens. As a result, considerable interest has been directed to the use of plants as alternatives to synthetic antioxidants in processed foods (Maisuthisakul et al. 2007). Essential oils (EOs) are volatile, natural, complex compounds characterized by a strong odor and are formed by aromatic plants as secondary metabolites. Since the middle ages, EOs have been widely used for bactericidal, virucidal, fungicidal, antiparasitical, insecticidal, medicinal and cosmetic applications (Bakkali et al. 2008). In the last decades, EOs and various extracts of plants have attracted great interest as they are derived from natural products. They have been screened for their potential use as alternative remedies for the treatment of many infectious diseases and in the preservation of foods from the toxic effects of oxidants (Kelen and Tepe 2008). The genus Thymus (Lamiaceae) is represented in Turkey by 39 species and 59 taxa, and the ratio of endemism in the genus is 53% (Karaman et al. 2001).Various Thymus species are aromatic plants of the Mediterranean flora. They are commonly used as spices and as remedies in traditional medicine. They are also reported to possess some biological effects such as antispasmodic, antibacterial, antiviral, expectorant and antioxidant activities (Ismaili et al. 2004). Thymus cappadocicus Boiss. is an aromatic and medicinal plant. It is normally located from 1,000 to 1,800 m above sea level and grows on open calcareous ground. The plant is a branched woody shrub with leaves that are linear with revolute margins, and the inflorescence is few flowered with white to lilac corolla (Davis 1982). The composition of the EO (Baser et al. 1996; Bouchra et al. 2003; Dorman et al. 2003; Bounatirou et al. 2007; Hazzit et al. 2009; Jordán et al. 2009), the antioxidant activity (Dorman et al. 2003; Ismaili et al. 2004; Bounatirou et al. 2007; Hazzit et al. 2009; Jordán et al. 2009; Orhan et al. 2009) and the antimicrobial activity (Karaman et al. 2001; Alzoreky and Nakahara 2003; Sagdic et al. 2009) of various Thymus species have been studied extensively. However, as far as our literature survey could ascertain, the antioxidant and antimicrobial activities of T. cappadocicus have not previously been published. In this study, we examined the chemical composition of T. cappadocicus EO using gas chromatography/mass spectrometry (GC/MS) and tested the antioxidant and antimicrobial activities of its EO and extracts.

MATERIALS AND METHODS Chemicals Folin–Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), sodium carbonate, gallic acid, ascorbic acid, nutrient agar, nutrient broth, malt extract agar and malt extract broth were purchased from Darmstadt, Germany. The other 606

chemicals and solvents used in this experiment were analytical grade, and also purchased from Merck.

Plant T. cappadocicus Boiss. was collected from Sivas, the inner Anatolian region of Turkey, in July 2009 (39o 31.017 N 36o 59.467 E, 1,380 m). Plants were collected during their flowering season. They were identified by senior taxonomist Prof. Dr. Ahmet Aksoy from Erciyes University’s Department of Biology. The voucher specimens were deposited at the Herbarium of the Department of Biology, Erciyes University, Kayseri, Turkey (Voucher No.: AAksoy 2357).

Extraction The dried aerial parts of the plant were crushed in a coffee grinder for 2 min at room temperature. At 15 s intervals, the process was stopped for 15 s to avoid overheating the sample. Powdered plant samples (10 g) were separately extracted using a Soxhlet type extractor with 100 mL methanol, ethanol and water. Thereafter, the extracts were filtered through Whatman No. 1 filter paper and evaporated to dryness in a vacuum at 40C with a rotary evaporator (Rotavator, Buchi, Switzerland; T < 40C). After determining the yield, the prepared extracts were stored at 4C until further analysis. The extracts obtained above indicated that solvents were expressed as methanol extract (ME), ethanol extract (EE) and water extract (WE), respectively.

Isolation of EO The air-dried and ground aerial parts of the plants were subjected to steam distillation for 3 h using a Clevenger-type apparatus. The EO obtained was dried over anhydrous sodium sulfate and, after filtration, stored at 4C until tested and analyzed. Yield was found as 3.3% (v/w).

GC/MS Analysis Conditions The composition of the volatile constituents was determined by GC/MS/quadrupole detection analysis using a Shimadzu QP 5050 system (Shimadzu, Duisburg, Germany) fitted with an FFAP (polyethylene glycol + 2-nitroterephthalate) capillary column (50 m ¥ 0.32 mm i.d., film thickness 1.2 mm). The detector and injector temperatures were set at 250 and 240C, respectively. The temperature of the column was held at 120C for 1 min, then increased at 2C/min to 220C and held for 20 min. Helium was used as a carrier gas at a flow rate of 10 psi (split 1:10). The injection volume of each sample was 1 mL. The ionization energy was set at 70 eV. Qualitative analysis was based on a comparison of retention times and mass spectra (Wiley, Nist and Tutor Libraries). The composi-



tion (%) of the EO was computed from the GC peak areas without using any correction factors (Sagdic et al. 2009).


Radical scavenging activity was expressed as a percentage inhibition of the DPPH radical and was calculated by the following equation:

Inhibition% = ([ Ablank − Asample ] Ablank ) × 100

Determination of Total Phenolics The total phenolic contents of the plant extracts were estimated by a colorimetric assay based on procedures described by Singleton and Rossi (1965). In short, a 40 mL aliquot of plant extracts dissolved in the same solvent was pipetted into a test tube containing 2.4 mL of distilled water. After mixing the contents, 200 mL of Folin–Ciocalteu’s phenol reagent and 600 mL of a saturated sodium carbonate solution (20% Na2CO3) were added. The contents were vortexed for 15 s and then left to stand at room temperature for 2 h. Absorbance measurements were recorded at 765 nm using a spectrophotometer (Shimadzu 1240, Kyoto, Japan) and gallic acid was used in the construction of the standard curve. Estimation of the phenolic content was carried out in triplicate. The results were given as mean values and expressed as mg of gallic acid equivalents (GAE)/g of dry extract.

Determination of Antioxidant Activity Phosphomolybdenum Assay. The antioxidant activities of the plant extracts were determined by the phosphomolybdenum method of Prieto et al. (1999). About 0.4 mL of the plant extract (1 mg/mL) was mixed with 4 mL of the reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a water bath at 95C for 90 min. After the samples had cooled to room temperature, the absorbance of the green phosphomolybdenum complex was measured at 695 nm. In the case of the blank, 0.4 mL of solvent was used in place of the sample. Antioxidant activity was determined using a standard curve with ascorbic acid solutions as the standard. The mean of three readings was used, and the reducing capacities of the extracts were expressed as mg of ascorbic acid equivalents (AAE)/g extract. DPPH Assay. The hydrogen atom or electron donation abilities of the plant extracts were measured by bleaching the purple-colored DPPH methanol solution. This spectrophotometric assay uses the stable radical DPPH as a reagent (Lee et al. 1998). About 50 mL of various concentrations (0.25– 5 mg/mL) of the plant extract in the same solvent were added to 1 mL of 0.1 mM DPPH methanol solution. After a 30 min incubation period at room temperature, absorbance was read against a blank at 517 nm. The IC50 (the concentration required to scavenge 50% DPPH free radicals) values of the plant extracts were determined graphically. The same procedure was repeated with BHT as a positive control. The measurements were performed in triplicate, and the results were averaged.

where Ablank is the absorbance of the control reaction (containing all reagents except the test compound) and Asample is the absorbance of the test compound. b-Carotene Bleaching Assay. The extracts’ ability to inhibit the bleaching of the b-carotene–linoleic acid emulsion was determined (Cao et al. 2009). b-Carotene (10 mg) was dissolved in 10 mL of chloroform (CHCl3). An aliquot (0.2 mL) of this solution was added to a boiling flask containing 20 mg of linoleic acid and 200 mg of Tween 40. The chloroform was removed using a rotary evaporator at 40C for 5 min. Distilled water (50 mL) was slowly added to the residue and mixed vigorously to form an emulsion. The emulsion (5 mL) was added to a tube containing 0.2 mL of the EO or the extract solution. The test emulsion was incubated in a water bath at 50C for 2 h, at which point the absorbance was measured at 470 nm. In the negative control, the EO or extracts were substituted with an equal volume of ethanol. BHT and BHA were used as positive controls.

Determination of Antimicrobial Activity Microorganisms. The following microorganisms obtained from the Department of Food Engineering in Erciyes University were used in this study: Aeromonas hydrophila ATCC 7965, Bacillus brevis FMC 3, Bacillus cereus RSKK 863, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Klebsiella pneumoniae FMC 5, Listeria monocytogenes 1/2B, Morganella morganii, Proteus mirabilis BC 3624, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium NRRLE 4463, Staphylococcus aureus ATCC 29213, Yersinia enterocolitica ATCC 1501, Candida albicans ATCC 1223 and Saccharomyces cerevisiae BC 5461. Agar-well Diffusion Method. Antimicrobial activity assays of the extracts and EO were carried out using the agarwell diffusion method (Sagdic et al. 2009). Each microorganism was suspended in sterile nutrient broth. Test yeasts (C. albicans, S. cerevisiae) were suspended in malt extract broth. Suspensions of microorganisms adjusted to 106–107 colony-forming units (cfu)/mL were placed in flasks containing 25 mL of sterile nutrient or malt extract agar at 45C. The mix was poured into Petri plates (9 cm in diameter). The agars were then allowed to solidify at 4C for 1 h. The wells (4 mm in diameter) were cut from the agar. The extracts were prepared at 30 mg/mL concentrations in the same solvent


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and 50 mL of the extract solutions and EO were then applied to the wells. Absolute methanol, absolute ethanol and water without herb extract were used as a control. Y. enterocolitica, C. albicans and S. cerevisiae were incubated at 25C for 24–48 h in an inverted position. The other microorganisms were incubated at 37C for 18–24 h. At the end of the incubation period, all plates were examined for any zones of growth inhibition, and the diameters of these zones were measured in millimeters. The standard antibiotics amoxicillin (AML-25 mg/disc), ampicillin (AMP-10 mg/disc), carbenicillin (CAR-100 mg/disc), chloramphenicol (C-30 mg/disc), erythromycin (E-15 mg/disc), gentamicin (CN-10 mg/disc), kanamycin (K-30 mg/disc), oxacillin (OX-1 mg/disc) and rifampicin (RD-5 mg/disc) (Oxoid) were used as positive controls. All the tests were performed in duplicate, and the results were presented as averages.

Determination of Minimum Inhibitory Concentration and Minimal Bactericidal Concentration The minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were also studied for the microorganisms, which were determined as sensitive to the extracts and EO in the agar-well diffusion assay. The inocula of the bacterial strains were prepared from 18 h broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity. First of all, the extracts and EO were dissolved in 10% dimethylsulfoxide. Then, they were diluted to the highest concentration (30 mg/mL for the extracts, 2,000 mg/mL for the EO) to be tested. Next, serial twofold dilutions were made of the extracts in a concentration range of 0.234–30 mg/mL and in a 7.8–2,000 mg/mL concentration range for the EO in 10-mL sterile test tubes containing nutrient broth. The MIC values of the extracts against bacterial strains were determined based on a micro-well dilution method (Sokmen et al. 2004). In brief, 96-well plates were prepared by dispensing 95 mL of nutrient broth and 5 mL of the inocula into each well. About 100 mL of the extracts and EO stock solutions were added into the first wells. Then, 100 mL of their serial dilutions were transferred into six consecutive wells. The last well, containing 195 mL of nutrient broth without compound and 5 mL of the inocula on each strip, was used as a negative control. The final volume in each well was 200 mL. The plate was covered with a sterile plate sealer. The contents of each well were mixed on a plate shaker at 300 rpm for 20 s and then incubated at 37C (25C for yeasts) for 24 h. The MIC was defined as the lowest concentration of the sample that prevented visible growth. MBCs were determined by subculturing (10 mL) from each negative tube and from the positive growth control. MBC was defined as the lowest concentration yielding negative subcultures or only one colony. Each assay in this experiment was repeated twice. 608

Statistical Analysis The antioxidant and antimicrobial results obtained were stated as mean ⫾ standard deviation. Analysis of variance (ANOVA) was performed by ANOVA procedures (SPSS 10.0 for Windows; SPSS Inc., Chicago, IL). Percentage data were transformed using arcsine ✓¥ before ANOVA. Means were separated at the 5% significance level by the least significant difference test. Bivariate correlations were analyzed by Pearson’s test using SPSS 10.0 for Windows.

RESULTS AND DISCUSSION This study determined the antioxidant and antimicrobial activities of the extracts (ME, EE and WE) and EO of T. cappadocicus. The percent yields of ME, EE and WE were 39.21, 16.25 and 16.36% (w/w), respectively. The percent yield of EO was 3.3% (v/w). The results of GC/MS analysis of the T. cappadocicus oil obtained by hydrodistillation are shown in Table 1. The chromatographic analyses resulted in the identification of eight components, representing 99.87% of the oils. The main compounds detected in the oil were thymol (70.82%), p-cymene (9.52%), g-terpinene (9.27%), carvacrol (4.65%), borneol (1.75%), a-terpinene (1.44%), myrcene (1.30%) and terpineol-4 (1.12%). To the best of our knowledge, many studies have been conducted on the chemical composition of the oils isolated from various Thymus species (Karaman et al. 2001; Hudaib et al. 2002; Bouchra et al. 2003; Sokmen et al. 2004; Tepe et al. 2005; Bounatirou et al. 2007; Hazzit et al. 2009; Jordán et al. 2009). However, only one study examined the EO composition of T. cappadocicus. The EOs of two varieties of T. cappadocicus were previously examined by Tabanca et al. (2000), who determined that 1,8-cineole, thymol, borneol, caryophyllene oxide and b-caryophyllene were its major compounds. In accordance with our results, Bouchra et al. (2003) found that Thymus glandulosus oil was TABLE 1. COMPOSITION OF THYMUS CAPPADOCICUS ESSENTIAL OIL Compound

RT†

%

Myrcene* a-Terpinene g-Terpinene Cymene Terpineol-4 Borneol Thymol Carvacrol

15.8 17.1 21.0 22.6 44.3 50.5 76.8 78.4

1.30‡ 1.44 9.27 9.52 1.12 1.75 70.82 4.65

* Compounds listed in order of elution from an FFAP mass spectrometry column. † Retention time (as minutes). ‡ The percentage composition was computed from the gas chromatography peak areas.



TABLE 2. THE YIELDS, TOTAL PHENOLIC CONTENTS AND TOTAL ANTIOXIDANT ACTIVITIES OF METHANOL, ETHANOL AND WATER EXTRACTS OBTAINED FROM THYMUS CAPPADOCICUS


Extracts

Yield (%)

Total phenolic content (mg GAE/g dry extract)

Total antioxidant activity (mg AAE/g dry extract)

Methanol Ethanol Water

39.21 16.25 16.36

108.43 ⫾ 1.3a* 51.68 ⫾ 0.3b 13.35 ⫾ 0.2c

262.51 ⫾ 0.3a 60.95 ⫾ 0.4b 45.53 ⫾ 0.0c

* Means followed by the same letter in a column are not significantly different at P = 0.05 (analysis of variance followed by least significant difference test). Values expressed are mean ⫾ standard deviation of three experiments. Total phenolic content expressed as gallic acid equivalent (GAE); total antioxidant activity expressed as ascorbic acid equivalent (AAE).

thymol rich (43.2%), while Bounatirou et al. (2007) and Karaman et al. (2001) reported that carvacrol was the predominant compound in Thymus capitatus and Thymus revolutus, respectively. Hudaib et al. (2002) evaluated seasonal variations in the composition of oil obtained from Thymusvulgaris harvested at different periods during the plant’s vegetative and life cycles, and they observed that thymol was the main compound. Also, the profile of the oil components from T. vulgaris was similar to that of T. cappadocicus in almost all compounds (myrcene, p-cymene, g-terpinene, borneol, carvacrol) but at different concentrations. In Sokmen et al.’s (2004) report, thymol (36.5%), carvacrol (29.8%), p-cymene (10.0%) and g-terpinene (6.3%) were identified as the main components in Thymus spathulifolius. These observations are partly in accordance with our results. It is noteworthy that the composition of EOs depends on various factors, particularly on climatic and environmental conditions (Tepe et al. 2005; Ložiene et al. 2007). In general, the antioxidant and radical scavenging properties of plant extracts are associated with the presence of phenolic compounds that possess the ability to donate hydrogen to the radical. A great number of simple phenolic compounds, as well as flavonoids, can act as antioxidants. However, their antioxidant properties depend on some important structural prerequisites, particularly on the number and arrangement of hydroxyl groups, the extent of structural conjugation, and the presence of electrondonating and electron-accepting subsistent on the ring structure (Miliauskas et al. 2005; Ložiene et al. 2007). The total phenolic contents of all T. cappadocicus extracts are presented in Table 2. The statistical differences among the extracts’ total phenolic contents were important (P < 0.05). The phenolic content of T. cappadocicus was highest in the ME at 108.43 ⫾ 1.3 mg GAE/g extract. The ME value was followed by that of EE (51.68 ⫾ 0.3 mg GAE/g extract) and WE (13.35 ⫾ 0.2 mg GAE/g extract). To the best of our knowledge, the total phenolic contents of T. cappadocicus are reported here for the first time. Jordán et al. (2009) reported that the total phenolic contents of Thymus zygis ssp. gracilis methanolic extracts ranged from 122.2 ⫾ 19.3 to 108.5 ⫾ 19.2 mg GAE/g dry plant. The total phenolic content of Thymus argaeus was found to be 83.31 ⫾ 0.59 mg GAE/g (Sagdic et al. 2009).

The possible antioxidant activities of ME, EE, WE and the EO of T. cappadocicus were examined using three complementary test systems, namely phosphomolybdenum assay, b-carotene bleaching assay and DPPH free radical scavenging assay. The extracts’ total antioxidant activities measured by phosphomolybdenum assay are given in Table 2. As can be seen from Table 2, the extracts of T. cappadocicus had strong total antioxidant activity. There were statistical differences among the total antioxidant activities of the extracts tested (P < 0.05). The total antioxidant activities of T. cappadocicus were estimated as 262.51 ⫾ 0.3, 60.95 ⫾ 0.3 and 45.53 ⫾ 0.0 mg AAE/g dry extract in ME, EE and WE, respectively. The free radical scavenging capacities of all the extracts and EO measured at different concentrations (0.25–5 mg/mL) by DPPH assay are shown in Fig. 1. To evaluate the free radical scavenging activities of all the extracts and EO, their inhibition rates were compared with that of the synthetic antioxidant BHT. The extracts and EO exhibited a concentrationdependent DPPH radical scavenging activity. Significant radical scavenging activity was evident in all the tested concentrations of T. cappadocicus extracts and EO. At 2 mg/mL, the percent inhibition rates of ME, EE, WE, EO and BHT were determined as 87.04, 87.22, 59.21, 48.14 and 92.15%, respectively. When compared with BHT, the free radical scavenging activities of ME and EE of T. cappadocicus were similar to that of BHT. The IC50 value is the amount of extract providing 50% inhibition of the DPPH radical. A lower IC50 value reflects better protective action of the extracts and EO. The lowest IC50 value of T. cappadocicus was determined in ME as 15.89 mg/mL. The highest IC50 value was determined in the EO as 72.77 mg/mL (P < 0.05) (Table 3). The ME, EE, WE and EO of T. cappadocicus showed strong antioxidant activities. ME was more effective in free radical scavenging activity and phosphomolybdenum assay. ME’s greater effectiveness may be due to more hydrogen donating components being extracted by methanol. The antioxidant activities of various Thymus species have been previously studied (Dorman et al. 2003; Ismaili et al. 2004; Miguel et al. 2004; Sokmen et al. 2004; Tepe et al. 2005; Bounatirou et al. 2007; Ložiene et al. 2007; Hazzit et al. 2009; Jordán et al. 2009; Orhan et al. 2009; Sagdic et al. 2009). However, to the best of


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100 90

Scavenging (%)

80 70 60 50 40 30 20 10 0 0.25

0.5 ME

1 2 3 Concentration (mg/mL) EE

4

WE

EO

our knowledge, no studies have examined the antioxidant activity of T. cappadocicus. When compared with other data, the DPPH free radical scavenging activities of T. cappadocicus extracts and EO were significantly higher than that of the WE of T. vulgaris (IC50 = 382.4 ⫾ 28.3 mg/mL) (Dorman et al. 2003). Also, Hazzit et al. (2009) showed that the two new chemotypes of Thymus algeriensis were not, or only slightly, active against the DPPH radical. Similarly, the radical scavenging activities (IC50 = 3.7–7.4 mg/mL) of T. zygis ssp. gracilis, cultivated at different watering levels, were lower than that of T. cappadocicus (Jordán et al. 2009). It is has been reported that the extracts of five chemotypes of Thymus pulegioides were strong DPPH free radical scavengers (Ložiene et al. 2007). Orhan et al. (2009) found that the ethyl acetate and EEs of Thymus praecox subsp. caucasicus exerted a significant DPPH scavenger effect with 87.80 and 85.42% inhibition at 2 mg/ mL, respectively.

TABLE 3. EFFECTS OF THYMUS CAPPADOCICUS EXTRACTS, ESSENTIAL OIL AND POSITIVE CONTROLS ON DPPH AND b-CAROTENE/LINOLEIC ACID SYSTEMS Extracts Methanol Ethanol Water Essential oil BHT BHA

DPPH IC50 (mg/mL) d

15.89 * 24.52c 55.28b 72.77a 7.40e –

b-Carotene bleaching (I%) 63.19d 56.90d 80.38bc 70.70c 84.26b 94.33a

* Means followed by the same letter in a column are not significantly different at P = 0.05 (analysis of variance followed by least significant difference test). DPPH, 2,2-diphenyl-1-picrylhydrazyl; BHT, butylated hydroxytoluene; BHA, butylated hydroxyanisole.

610

5 BHT

FIG. 1. 2,2-DIPHENYL-1-PICRYLHYDRAZYL RADICAL SCAVENGING ACTIVITIES (%) OF THYMUS CAPPADOCICUS ME, methanol extract; EE, ethanol extract; WE, water extract; EO, essential oil; BHT, butylated hydroxytoluene.

The ME of Thymus caramanicus was also tested for its free radical scavenging activity, and the polar subfraction of the ME was found to possess radical scavenging activity (IC50 = 43.0 mg/mL) (Safaei-Ghomi et al. 2009). In another study, the polar subfraction of the ME of T. spathulifolius showed DPPH radical scavenging activity with an IC50 of 16.15 mg/mL which is lower than that of the T. cappadocicus ME (Sokmen et al. 2004). The free radical scavenging activities of the EOs of Thymus sipyleus subsp. sipyleus var. rosulans and T. sipyleus subsp. sipyleus var. sipyleus (IC50 = 220 ⫾ 0.5 and 2,670 ⫾ 0.5 mg/mL, respectively) were significantly lower than that of T. cappadocicus (Tepe et al. 2005). Miguel et al. (2004) stated that the EOs of Thymus caespititius, Thymus camphoratus and Thymus mastichina showed strong antioxidant capacities. The antioxidant properties of thymol, carvacrol and g-terpinene have been previously reported. Therefore, the antioxidant activity of an EO could be attributed to the high contents of components present in the oil (Sokmen et al. 2004). The present study represents the first report of the total antioxidant activities of T. cappadocicus extracts and EO using phosphomolybdenum assay. In the b-carotene/linoleic acid bleaching assay, the oxidation of linoleic acid generates peroxyl radicals, thereby producing free radicals, which will oxidize the highly unsaturated b-carotene resulting in a bleaching effect.The presence of antioxidants in the extract minimizes the oxidation of b-carotene by hydroperoxides. An extract that possesses an antioxidant effect may prevent the oxidation of lipid components within cell membranes (Cao et al. 2009; Mariod et al. 2009). In b-carotene/linoleic acid assay, the oxidation of linoleic acid was effectively inhibited by T. cappadocicus extracts and EO (Table 3). WE and EO were found to possess maximum antioxidant activities of 80.38 and 70.70%, respectively. The inhibition rate of WE was close to the synthetic antioxidant BHT (84.26%) (P < 0.05).



Safaei-Ghomi et al. (2009) reported that the nonpolar subfraction of the T. caramanicus ME showed high inhibition (84.4%), which is in agreement with our results. In another report, the inhibition values of linoleic acid oxidation were calculated as 92 and 89% for the oil and the polar subfraction of T. spathulifolius, respectively (Sokmen et al. 2004). On the other hand, it has been reported that the oxidation of linoleic acid was effectively inhibited by T. sipyleus subsp. sipyleus var. rosulans (92.0%), while the var. sipyleus oil showed no activity (Tepe et al. 2005). Bivariate correlations were analyzed by Pearson’s test using SPSS 10.0 for Windows. It was observed that a correlation existed between the total antioxidant activities and total phenolic contents of T. cappadocicus extracts (r = 0.940). In addition, a correlation was observed between the total phenolic contents and free radical scavenging activities of these extracts (r = 0.911). However, such a correlation was not found between total phenolic contents and b-carotenelinoleic bleaching inhibition activities (r = -0.597). The in vitro antimicrobial activities of the EO and extracts obtained from T. cappadocicus against certain microorganisms were investigated, and their activity potentials were assessed by the inhibition zone diameter, MIC and MBC values (Tables 4 and 5). In the present study, food pathogens were selected as target microorganisms for the preliminary antimicrobial screening of the EO and extracts of T. cappadocicus. According to the results given in Tables 4 and 5, the T. cappadocicus EO exhibited great potential antimicrobial activity against the 13 bacteria and two yeasts tested. Among the extracts, EE was the most effective against the microorganisms tested. The WE of T. cappadocicus exhibited no activity against the microorganisms tested. ME was also found to be effective against five out of the 13 bacterial strains examined, but no activity was evident against the yeasts tested. In this study, the antimicrobial activities of extracts and EO were compared with standard antibiotics (Table 4). As can be seen in Table 4, P. aeruginosa ATCC 27853 and B. cereus RSKK 863 were the most sensitive bacteria to ME. P. aeruginosa ATCC 27853 and B. brevis FMC 3 were the most sensitive bacteria while E. coli ATCC 25922 was the most resistant bacterium to EE. B. cereus RSKK 863 and A. hydrophila ATCC 7965 were the most sensitive bacteria to T. cappadocicus EO while the most resistant bacterium was K. pneumoniae FMC 5. The maximal inhibition zones for bacterial strains and yeasts, which were sensitive to the ME, EE and T. cappadocicus EO, ranged from 9.5–14.5, 7–13.5 and 13–54 mm, respectively (Table 4). Based on these results, it is possible to conclude that T. cappadocicus EO displays stronger activity and has a broader spectrum than all the other extracts. The significant antimicrobial effects, expressed as MIC and MBC (MFC = Minimum fungicidal concentration for C. albicans and S. cerevisiae) of the extracts and EO against the microorganisms, are shown in Table 5. The EO was most active with MIC


values ranging from 0.03 to 1.00 mg/mL. Among the extracts tested, EE showed very strong activity against the examined microorganisms with MIC values ranging from 1.87 to 7.50 mg/mL. The EE MIC values were followed by ME (1.87– 15.00 mg/mL). The MBC values were in the range of 7.5– 15.00 mg/mL, 1.87–7.50 mg/mL and 0.25–1.00 mg/mL for ME, EE and EO, respectively. The antimicrobial properties of EOs and extracts obtained from various Thymus species have been reported in numerous studies (Karaman et al. 2001; Rasooli and Mirmostafa 2002; Alzoreky and Nakahara 2003; Bouchra et al. 2003; Ismaili et al. 2004; Sokmen et al. 2004; Dob et al. 2006; Rasooli et al. 2006; Bounatirou et al. 2007; Ložiene et al. 2007; Sagdic et al. 2009). However, the antimicrobial properties of T. cappadocicus have not been reported before. The antibacterial activities of Thymus serpyllum extracts were tested against B. cereus, E. coli, Salmonella infantis, L. monocytogenes and S. aureus, and were found to possess an antibacterial effect with an MIC of 660– > 2.640 mg/mL (Alzoreky and Nakahara 2003). In a previous study, it was stated that B. cereus, Micrococcus luteus, Staphylococcus epidermidis and S. aureus were the most sensitive to all extracts of T. pulegioides, whereas E. coli, S. typhimurium and Enterobacter aerogenes remained resistant. The antibacterial activity of an extract depends on the plant chemotype, extract preparation, solvent used and, finally, on the sensitivity of the bacteria (Ložiene et al. 2007). However, it was determined that Thymus satureioides extracts did not show any antibacterial effect against four standard aerobic bacteria strains (Ismaili et al. 2004). The EOs of various Thymus species were also found to possess antifungal activities (Bouchra et al. 2003; Rasooli et al. 2006). Recently, Dob et al. (2006) found that T. algeriensis EO exhibited significant in vitro antimicrobial activity against B. subtilis (MIC = 0.5 mL/mL), as well as against the two yeasts and two filamentous fungi tested (MIC = 0.5 and 1.0 mL/mL). Hazzit et al. (2009) showed that the EOs of T. algeriensis, Thymus pallescens and Thymusdréatensis possessed antimicrobial activity against B. cereus, L. monocytogenes, Salmonella sp., S. aureus, Helicobacter pylori and C. albicans in the range of 8.00–34.67 mm inhibition zones. T. revolutus EO was tested for its antimicrobial effect against 11 bacteria and four fungi, and it was found to possess significant antibacterial and antifungal activity, which is in keeping with our results (Karaman et al. 2001). The antibacterial properties of Thymus pubescens and T. serpyllum EOs were studied, and the oils were found to possess bactericidal properties (Rasooli and Mirmostafa 2002). According to the results shown in Sokmen et al.’s (2004) report, the EO, and the nonpolar and polar subfractions of the T. spathulifolius ME had great potential antimicrobial activity. Sagdic et al. (2009) studied the antimicrobial activities of T. argaeus extract and EO against 13 bacteria and two yeasts, and it was determined that the most sensitive microorganisms to the EO


611

612 – –

8.0 ⫾ 0.0 7.0 ⫾ 0.0

– –

– – – – –

13.5 ⫾ 0.7 13.0 ⫾ 1.4 9.0 ⫾ 0.0 12.0 ⫾ 0.0 10.5 ⫾ 0.7

– – – –

10.0 ⫾ 1.4 14.5 ⫾ 0.7 – 9.5 ⫾ 0.7 –

– – –

WE

13.0 ⫾ 0.0 8.5 ⫾ 0.7 10.5 ⫾ 0.7 9.0 ⫾ 0.0 9.5 ⫾ 0.7 13.5 ⫾ 0.7 9.0 ⫾ 0.0 11.0 ⫾ 0.0

EE

9.5 ⫾ 0.7* – – – – 11.0 ⫾ 0.0 – –

ME

32.5 ⫾ 0.7 39.5 ⫾ 0.7

42.5 ⫾ 0.7 54.0 ⫾ 0.0 18.0 ⫾ 0.0 21.5 ⫾ 0.7 15.5 ⫾ 0.7

44.0 ⫾ 0.0 19.0 ⫾ 1.4 15.0 ⫾ 0.0 13.0 ⫾ 0.7 21.0 ⫾ 1.4 33.0 ⫾ 0.0 16.5 ⫾ 2.1 20.5 ⫾ 0.7

EO

– –

12.0 ⫾ 0.0 34.0 ⫾ 0.0 25.0 ⫾ 0.0 33.0 ⫾ 0.0 17.0 ⫾ 0.0

33.0 ⫾ 0.0† 12.0 ⫾ 0.0 – 16.0 ⫾ 0.0 31.0 ⫾ 0.0 30.0 ⫾ 0.0 29.0 ⫾ 0.0 13.0 ⫾ 0.0

AML

Antibiotics (mL)

– –

8.0 ⫾ 0.0 31.0 ⫾ 0.0 24.0 ⫾ 0.0 28.0 ⫾ 0.0 16.0 ⫾ 0.0

27.0 ⫾ 0.0 6.5 ⫾ 0.0 – 14.0 ⫾ 0.0 26.0 ⫾ 0.0 25.0 ⫾ 0.0 24.0 ⫾ 0.0 8.0 ⫾ 0.0

AMP

– –

20.0 ⫾ 0.0 15.0 ⫾ 0.0 15.0 ⫾ 0.0 15.0 ⫾ 0.0 12.0 ⫾ 0.0

13.0 ⫾ 0.0 7.0 ⫾ 0.0 6.5 ⫾ 0.0 11.0 ⫾ 0.0 13.0 ⫾ 0.0 12.0 ⫾ 0.0 10.0 ⫾ 0.0 14.0 ⫾ 0.0

K

– –

20.0 ⫾ 0.0 21.0 ⫾ 0.0 25.0 ⫾ 0.0 25.0 ⫾ 0.0 15.0 ⫾ 0.0

18.0 ⫾ 0.0 17.0 ⫾ 0.0 11.0 ⫾ 0.0 13.0 ⫾ 0.0 19.0 ⫾ 0.0 15.0 ⫾ 0.0 22.0 ⫾ 0.0 17.0 ⫾ 0.0

C

– –

9.0 ⫾ 0.0 38.0 ⫾ 0.0 24.0 ⫾ 0.0 31.0 ⫾ 0.0 13.0 ⫾ 0.0

35.0 ⫾ 0.0 6.5 ⫾ 0.0 18.0 ⫾ 0.0 12.0 ⫾ 0.0 30.0 ⫾ 0.0 31.0 ⫾ 0.0 26.0 ⫾ 0.0 9.0 ⫾ 0.0

CAR

– –

16.0 ⫾ 0.0 11.0 ⫾ 0.0 12.0 ⫾ 0.0 13.0 ⫾ 0.0 7.0 ⫾ 0.0

6.5 ⫾ 0.0 8.0 ⫾ 0.0 12.0 ⫾ 0.0 8.0 ⫾ 0.0 9.0 ⫾ 0.0

–

8.5 ⫾ 0.0 9.0 ⫾ 0.0

CN

– –

22.0 ⫾ 0.0 18.0 ⫾ 0.0 20.0 ⫾ 0.0 23.0 ⫾ 0.0 12.0 ⫾ 0.0

20.0 ⫾ 0.0 – – 11.0 ⫾ 0.0 – – – 7.0 ⫾ 0.0

E

– –

– 20.0 ⫾ 0.0 19.0 ⫾ 0.0 – –

15.0 ⫾ 0.0 – – – – – – –

OX

– –

10.0 ⫾ 0.0 17.0 ⫾ 0.0 18.0 ⫾ 0.0 24.0 ⫾ 0.0 11.0 ⫾ 0.0

17.0 ⫾ 0.0 10.0 ⫾ 0.0 10.0 ⫾ 0.0 11.0 ⫾ 0.0 10.0 ⫾ 0.0 13.0 ⫾ 0.0 12.0 ⫾ 0.0 12.0 ⫾ 0.0

RD

* Values expressed are mean ⫾ standard deviation of two experiments, inhibition zones include diameter of hole (5 mm), sample amount 50 mL. † Inhibition zones include diameter of disc (6 mm). Amoxicillin (AML-25 mg/disc), Ampicillin (AMP-10 mg/disc), Carbenicillin (CAR-100 mg/disc), Chloramphenicol (C-30 mg/disc), Erythromycin (E-15 mg/disc), Gentamicin (CN-10 mg/disc), Kanamycin (K-30 mg/disc), Oxacillin (OX-1 mg/disc), Rifampicin (RD-5 mg/disc). –: Not active. EE, ethanol extract; EO, essential oil; ME, methanol extract; WE, water extract.

Gram (-) Aeromonas hydrophila Escherichia coli Morganella morganii Klebsiella pneumoniae Proteus mirabilis Pseudomonas aeruginosa Salmonella typhimurium Yersinia enterocolitica Gram (+) Bacillus brevis Bacillus cereus Bacillus subtilis Listeria monocytogenes Staphylococcus aureus Yeasts Candida albicans Saccharomyces cerevisiae

Microorganisms

Extracts and essential oil

TABLE 4. ANTIMICROBIAL ACTIVITIES OF EXTRACTS AND ESSENTIAL OIL OF THYMUS CAPPADOCICUS, AND SOME ANTIBIOTICS AS POSITIVE CONTROL

BIOACTIVITY OF THYMUS CAPPADOCICUS S. ALBAYRAK and A. AKSOY




TABLE 5. MICS AND MBCS OF THE THYMUS CAPPADOCICUS EXTRACTS AND ESSENTIAL OIL Methanol extract Microorganisms Gram (-) Aeromonas hydrophila Escherichia coli Morganella morganii Klebsiella pneumoniae Proteus Mirabilis Pseudomonas aeruginosa Salmonella typhimurium Yersinia enterocolitica Gram (+) Bacillus brevis Bacillus cereus Bacillus subtilis Listeria monocytogenes Staphylococcus aureus Yeasts Candida albicans Saccharomyces cerevisiae

MIC (mg/mL)

Ethanol extract

Essential oil

MBC (mg/mL)

MIC (mg/mL)

MBC (mg/mL)

MIC (mg/mL)

MBC (mg/mL)

15.00 – – – – ND – –

3.75 7.50 7.50 3.75 7.50 3.75 7.50 3.75

ND 7.50 7.50 3.75 7.50 ND 7.50 7.50

0.50 0.50 0.03 1.00 0.5 0.5 0.5 0.5

ND 1.00 1.00 1.00 0.50 1.00 0.50 1.00

– 15.00 –

ND 7.50 – ND –

1.87 1.87 3.75 3.75 3.75

ND 1.87 7.50 3.75 3.75

0.5 0.5 0.5 0.5 1.00

ND 0.50 1.00 1.00 1.00

– –

– –

7.50 7.50

ND ND

0.25 0.12

0.50 0.25

3.75 – – – – 3.75 – – 3.75 1.87

–: Not active. ND, not detected; MIC, minimal inhibitory concentration; MBC, minimum bactericidal concentration.

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