Food Control 15 (2004) 627–634

www.elsevier.com/locate/foodcont

The in vitro antimicrobial and antioxidant activities of the

essential oils and methanol extracts of endemic Thymus spathulifolius
Atalay Sokmen a,*, Medine Gulluce b,c, H. Askin Akpulat a, Dimitra Daferera d,
Bektas Tepe a, Moschos Polissiou d, M€unevver Sokmen e, Fikrettin Sahin f,c
a
Department of Biology, Faculty of Science and Literature, Cumhuriyet University, 58140 Sivas, Turkey
b
Department of Biology, Faculty of Art and Science, Atat€urk University, 25240 Erzurum, Turkey
c
Atat€urk University, Biotechnology Application and Research Center, 25240 Erzurum, Turkey
d
Laboratory of General Chemistry, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece
e
Department of Chemistry, Faculty of Science and Literature, Cumhuriyet University, 58140 Sivas, Turkey
f
Department of Plant Protection, Faculty of Agriculture, Atat€urk University, 25240 Erzurum, Turkey
Received 8 June 2003; received in revised form 15 October 2003; accepted 16 October 2003

Abstract
The present study was conducted to evaluate the in vitro antimicrobial and antioxidant properties of essential oil and methanol
extracts from a unique and endemic plant, Thymus spathulifolius (Hausskn. and Velen.). The antimicrobial test results showed that
the essential oil of T. spathulifolius strongly inhibited the growth of test microorganisms studied, except for 4 fungi species while
polar and non-polar subfractions of the methanol extract had moderate antibacterial, but not antifungal and anticandidal activity.
The antioxidative potential of the samples was evaluated using two separate methods, inhibition of free radical 2,2-diphenyl-1-
picrylhydrazyl (DPPH) and b-carotene–linoleic acid systems. The polar subfraction of the methanol extract was able to reduce the
stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) with an IC50 of 16.15 ± 0.5 lg/ml, which was lower than that of synthetic
antioxidant, BHT, (19.8 ± 0.5 lg/ml). Inhibition values of linoleic acid oxidation were calculated as 92% and 89% for the oil and the
polar subfraction, respectively. Gallic acid equivalent total phenolic constituent of the polar subfraction was 141.00 ± 0.90 lg/mg
(14.1%, w/w). The chemical composition of a hydrodistilled essential oil of T. spathulifolius was analyzed by a GC and GC/MS
system. A total of 28 constituents representing 99.2% of the oil were identified; thymol (36.5%), carvacrol (29.8%), p-cymene (10.0%)
and c-terpinene (6.3%) were the main components comprising 82.6% of the oil. Results presented here may suggest that the essential
oil and extracts of T. spathulifolius possess antimicrobial and antioxidant properties, and therefore, they can be used as a natural
preservative ingredient in food and/or pharmaceutical industry.

2003 Elsevier Ltd. All rights reserved.

Keywords: Thymus spathulifolius; Antimicrobial activity; Antioxidant activity

1. Introduction Van Beek, & Linssen, 1998). Also, increasingly adverse

drug reactions to the synthetic antibiotics and the
Synthetic antioxidants such as butylated hydroxy- increasing resistance of some pathogens to synthetic
anisole (BHA) and butylated hydroxytoluene (BHT) are antibiotics, has been another argument against the use
widely used as potential inhibitors of lipid peroxidation of these chemicals as therapeutics (Agnihotri & Vaidya,
and thereby stabilizing fat-containing food-stuffs. 1996; Friedman, Henika, & Mandrell, 2002).
However, due to their unstable and highly volatile nat- Commercial antimicrobial drugs/chemicals used
ure, they have frequently brought some questions about haphazardly in the treatment of many infectious diseases
their safety and efficiency ever since their first intro- has inevitably led multiple drug/chemical resistance
duction to the food industry (Dapkevicius, Venskutonis, in both human and plant pathogenic microorgan-
isms (Davis, 1994; Loper et al., 1991; Service, 1995).
Besides, food-borne diseases are still a major problem
*
Corresponding author. Tel.: +90-346-219-1010x2907; fax: +90-
in the World, even in well-developed countries (Mead
346-219-1186. et al., 1999). A variety of microorganisms also lead
E-mail address: asokmen@cumhuriyet.edu.tr (A. Sokmen). food spoilage that is encountered as one of the most
0956-7135/$ - see front matter
2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2003.10.005
628 A. Sokmen et al. / Food Control 15 (2004) 627–634

important matter concerning the food industry. So far, tems. The chemical composition of the essential oil was
many pathogenic microorganisms, such as Escherichia analysed by using GC and GC-MS.
coli, Staphylococcus aureus, Klebsiella pneumoniae, Lis-
teria monocytogenes and Campylobacter jejuni, Candida
spp., Zygosaccharomyces spp., Fusarium spp., Aspergil- 2. Materials and methods
lus spp., Rhizopus spp., and Penicillium spp. have been
reported as the causal agents of foodborne diseases and/ 2.1. Plant material
or food spoilage (Betts, Linton, & Betteridge, 1999;
Deak & Beuchat, 1996; Pitt & Hocking, 1997; Walker, Thymus spathulifolius plants at the flowering stage
1988). Raw and/or processed foods are open to con- were collected from nearby Yagbasan village, Sincan
tamination during the production, sale any distribution (1350 m), Sivas,Turkey. The taxonomic identification of
of the foods (Deak & Beuchat, 1996). Thus, at present, it plant materials was confirmed by a senior plant taxon-
is a necessity to use the chemical preservatives to prevent omist, H. Askin Akpulat, in Department of Biology,
the growth of food spoiling microbes in the food Cumhuriyet University, Sivas, Turkey. Due to its unique
industry (Sa €
gdıcß & Ozcan, 2003). Due to the economical nature, we conducted our research with a careful col-
impacts of spoiled foods and the consumer’s concerns lection, thereby using limited material, in order to avoid
over the safety of foods containing synthetic chemicals, giving damage to this species.
a lot of attention has been paid to naturally derived Collected plant materials were dried in the shade, the
compounds or natural products (Alzoreky & Nakahara, leaves of plants were separated from the stem, and
2003; Hsieh, Mau, & Huang, 2001). Recently, there has ground in a grinder with a 2 mm in diameter mesh. The
been considerable interest in extracts and essential oils voucher specimen has been deposited at the Herbarium
from aromatic plants with antimicrobial activities for of the Department of Biology, Cumhuriyet University,
controlling pathogens and/or toxin producing microor- Sivas-Turkey (CUFH-Voucher No: AA 3104).
ganisms in foods (Alzoreky & Nakahara, 2003; Soliman
& Badeaa, 2002; Valero & Salmeron, 2003). 2.2. Preparation of the methanol extracts
Evidently, members of the genus Thymus are such
natural sources, including a well-known species (Thymus The air-dried and finely ground sample was extracted
vulgaris) (thyme), antimicrobial and antioxidant pro- by using the method described previously (Sokmen,
perties of which have been extensively studied (Marino, Jones, & Erturk, 1999). The resulting extract (13.11%,
Bersani, & Comi, 1999; Reddy, Angers, Gosselin, & w/w) was suspended in water and partitioned with
Arul, 1998; Ryman, 1992; Schwarz, Ernst, & Ternes, chloroform (CHCI3 ) to obtain water-soluble (polar)
1996; Simandi et al., 2001). Reports concerning the (10.86%, w/w) and water-insoluble (non-polar, chloro-
antimicrobial and antioxidant nature of other Thymus formic) subfractions (2.25%, w/w), which were then
species are also numerous in the literature (Karaman, lyophilised and kept in the dark at +4 C until tested.
Digrak, Ravid, & Ilcim, 2001; Rasooli & Mirmostafa,
2002; Vardar-Unl€€ u et al., 2003; Youdim, Deans, & 2.3. Isolation of the essential oil
Finlayson, 2002).
The genus Thymus is represented by 38 species and The air-dried and ground herbal parts of plants col-
altogether 64 taxa; 24 of which are endemic in Turkey lected were submitted for 3 h to water-distillation using
and the East Aegean Islands (Davis, 1982). Thymus a British-type Clevenger apparatus (ILDAM Ltd., An-
spathulifolius is an endemic species, narrowly distributed kara-Turkey) (yield 3.74% v/w). The obtained essential
in Inner Anatolia. To the best of our knowledge, the oil was dried over anhydrous sodium sulphate and after
essential oil composition, antimicrobial and antioxidant filtration, stored at +4 C until tested and analysed.
properties of it have not been reported before. T. spa-
thulifolius is a rare and endemic species, narrowly dis- 2.4. GC and GC-MS analysis conditions
tributed in Sivas (Davis, 1982; IUCN, 2001).
The objectives of this study were (i) to investigate the GC analysis was performed on a Hewlett Packard
antimicrobial and antioxidant activity of the essential oil 5890 II gas chromatograph equipped with a FID and
and extracts from T. spathulifolius, and (ii) to determine HP-5ms capillary column (bonded and cross-linked 5%-
the chemical composition of its hydrodistilled essential phenyl-methylpolysiloxane 30 m · 0.25 mm i.d., film
oil by GC/MS. The antimicrobial activity was deter- thickness 0.25 lm). Injector and detector temperatures
mined by using agar well-diffusion, agar disc diffusion were set at 220 and 290 C, respectively. The oven tem-
and broth microdilution methods, while the antioxidant perature was held at 50 C for 3 min, then programmed
activity was evaluated using two separate methods, to 240 C at a rate of 3 C/min. Helium was the carrier
namely inhibition of free radical 2,2-diphenyl-1- gas, at a flow rate of 1 mL/min. Diluted samples (1/100 in
picrylhydrazyl (DPPH) and b-carotene–linoleic acid sys- acetone, v/v) of 1.0 lL were injected manually and in the
 A. Sokmen et al. / Food Control 15 (2004) 627–634 629

Table 1 belonging to 24 bacteria, 15 fungi and yeast species. The

Chemical composition of T. spathulifolius essential oil list of microorganisms used is given in Tables 2 and 3.
PeakNo RRIa Components Composition Microorganisms were provided by the Department of
(%) Clinical Microbiology, Faculty of Medicine and Plant
1 1129 a-Thujene 0.4 Diagnostic Laboratory, Faculty of Agriculture at
2 1144 a-Pinene 0.3
Atat€ urk University, Erzurum, Turkey. Identity of the
3 1176 Camphene 0.3
4 1259 3-Octanone 0.7 microorganisms used in this study was confirmed by
5 1267 b-Myrcene 0.9 microbial identification system (MIS) in Biotechnology
6 1290 3-Octanol 0.3 Application and Research Center at Atat€ urk University.
7 1311 a-Terpinene 0.9 MIS is an automated gas chromatography system with a
8 1325 p-Cymene 10.0
computer interface and software (Sherlock Microbial
9 1338 1,8-Cineole 1.2
10 1381 c-Terpinene 6.3 Identification System), with databases of FAME profiles
11 1399 cis-Sabinene hydrate 1.2 of yeast, fungi, bacteria and actinomycetes, which has
12 1427 Terpinolene 0.2 been developed by MIDI (Microbial ID, Newark, DE),
13 1445 trans-Sabinene hydrate 0.4 and used for microbial identification and characteriza-
14 1501 Camphor 0.1
tion for last two decades (Stead, 1988).
15 1535 Borneol 6.0
16 1544 Terpinen-4-ol 1.2
17 1556 Cymen-8-ol (para) 0.3 2.5.2. Disc-diffusion assay
18 1560 a-Terpineol 0.4 The dried plant extracts were dissolved in the same
19 1564 Dihydro carvone* 0.2 solvent (methanol) to a final concentration of 30 mg/ml
20 1617 Thymoquinone 0.3
and sterilized by filtration by 0.45 lm Millipore filters.
21 1621 Thymoquinone isomer 0.3
22 1663 Thymol 36.5 Antimicrobial tests were then carried out by disc diffu-
23 1672 Carvacrol 29.8 sion method (Murray, Baron, Pfaller, Tenover, &
24 1705 Thymol acetate 0.2 Yolke, 1995) using 100 ll of suspension containing 108
25 1715 Piperitenone oxide 0.1 CFU/ml of bacteria, 106 CFU/ml of yeast and 104 spore/
26 1718 Carvacrol acetate 0.1
ml of fungi spread on nutrient agar (NA), Sabouraud
27 1749 b-Caryophyllene 0.4
28 1843 Caryophyllene oxide 0.2 dextrose agar (SDA) and potato dextrose agar (PDA)
medium, respectively. The discs (6 mm in diameter) were
Total identified 99.2
impregnated with 10 ll of essential oil or the 30 mg/ml
*Correct isomer not identified. extracts (300 lg/disc) and placed on the inoculated agar.
a
RRI; Relative retention index on non-polar HP-5ms column in
Negative controls were prepared using the same solvents
reference to n-alkanes.
employed to dissolve the plant extracts. Ofloxacin (10
lg/disc), sulbactam (30 lg) + cefoperazona (75 lg) (105
splitless mode. Quantitative data were obtained elec- lg/disc) and/or netilmicin (30 lg/disc) were used as po-
tronically from FID area percent data. sitive reference standards to determine the sensitivity of
GC-MS analysis of the essential oil was performed one strain/isolate in each microbial species tested. The
under the same conditions with GC (column, oven inoculated plates were incubated at 37 C for 24 h for
temperature, flow rate of the carrier gas) using a Hewlett clinical bacterial strains, 48 h for yeast and 72 h for
Packard 5890 II gas chromatograph equipped with a fungi isolates. Plant associated microorganisms were
Hewlett Packard 5972 mass selective detector in the incubated at 27 C. Antimicrobial activity was evaluated
electron impact mode (70 eV). Injector and MS transfer by measuring the zone of inhibition against the test
line temperatures were set at 220 and 290 C, respec- organisms.
tively. The components were identified based on the
comparison of their relative retention time and mass 2.5.3. Micro-well dilution assay
spectra with those of standards, NBS75K library data of The minimal inhibitory concentration (MIC) values
the GC-MS system and literature data (Adams, 2001). were studied for the bacterial strains, being sensitive to
Alkanes were used as reference points in the calculation the essential oil and/or extracts in the disc diffusion
of relative retention indices (RRI). GC and GC-MS assay. The inocula of the bacterial strains were prepared
analysis results are given in Table 1. from 12 h broth cultures and suspensions were adjusted
to 0.5 McFarland standard turbidity. The essential oils
2.5. Antimicrobial activity and extracts were first dissolved in 10% dimethylsulf-
oxide (DMSO) and then diluted to the highest concen-
2.5.1. Microbial strains tration (500 lg/ml) to be tested, and then serial two-fold
Subfractions of the methanol extract and the essential dilutions were made in a concentration range from
oil were individually tested against a panel of microor- 7.8 to 500 lg/ml in 10 ml sterile test tubes contain-
ganisms including a total of 40 microbial cultures ing nutrient broth. MIC values of the extracts against
630 A. Sokmen et al. / Food Control 15 (2004) 627–634

Table 2
Antimicrobial activities of the essential oil and subfractions of the methanol extracts from Thymus spathulifolius against the bacterial strains tested
Test microorganisms The oil Subfractions of MeOH Extract Antibioticsa
CHCl3 H2 O
DDb MICc DDd MIC DD MIC DD MIC (max)
Acinetobacter baumanii-A8 19 250 14 125 – – 18 (OFX) 31.25
Bacillus macerans-A199 18 7.81 12 62.50 10 125 19 (OFX) 15.62
B. megaterium-M3 28 125 10 15.62 – – 9 (SCF) 15.62
B. subtilis-ATCC-6633 21 250 16 250 10 500 28 (OFX) 62.50
B. subtilis-A57 24 3.90 13 15.62 8 250 28 (OFX) 125
Brucella abortus A77 7 15.62 – – – – 12 (SCF) 62.50
Burkholdria cepacia A225 12 7.81 – – – – 22 (SCF) 125
Clavibacter michiganense-A227 17 7.81 12 250 – – 25 (SCF) 16.62
Enterobacter cloacae-A135 24 62.50 – – – – 20 (NET) 31.25
Enterococcus faecalis-ATCC-29122 21 62.50 14 125 10 250 18 (SCF) 31.25
Escherichia coli-A1 32 62.50 29 250 27 31.25 – (OFX) 62.50
Klebsiella pneumoniae-A137 17 250 – – – – 12 (OFX) 125
Proteus vulgaris-A161 24 125 – – – – 12 (OFX) 125
P. vulgaris-KUKEM1329 24 125 12 125 14 250 13 (OFX) 125
Pseudomonas aeruginosa-ATCC9027 24 125 14 250 8 250 22 (NET) 31.25
P. aeruginosa-ATCC-27859 27 125 10 6250 12 500 22 (NET) 15.62
P. syringae pv. tomato.-A35 9 250 – – – – 24 (OFX) 125
Salmonella enteritidis-ATCC-13076 24 250 17 500 16 250 27 (SCF) 62.50
Staphylococcus aureus-A215 23 125 14 7.81 14 250 22 (SCF) 31.25
S. aureus-ATCC-29213 21 250 10 250 14 250 22 (SCF) 62.50
Staphylococcus epidermis-A233 24 125 13 7.81 – – – (SCF) 15.62
Streptococcus pyogenes-ATCC-176 23 7.81 13 500 12 500 10 (OFX) 62.50
Streptococcus pyogenes-KUKEM-676 27 125 12 250 9 500 13 (OFX) 31.25
Xanthomonas campestris-A235 11 500 13 15.62 – – 20 (SCF) 31.25
a
OFX ¼ Ofloxacin (10 lg/disc); SCF ¼ sulbactam (30 lg)+cefoperazona (75 lg) (105 lg/disc) and NET ¼ Netilmicin, (30 lg/disc) (Oxoid);
MAX ¼ Maxipine (lg/mL) was used as reference antibiotic in micro well dilution assay (Sigma).
b
Inhibition zone in diameter (mm) around the discs impregnated with 10 ll of essential oil.
c
Minimal Inhibitory concentrations (as lg/ml).
d
Inhibition zone in diameter (mm) around the discs impregnated with extracts (300 lg/disc).

bacterial strains and Candida albicans isolates were study was screened twice against each organism. The
determined based on a micro-well dilution method as MIC was defined as the lowest concentration of the
previously described (Sahin et al., 2003). compounds to inhibit the growth of microorganisms.
In brief, the 96-well plates were prepared by dis-
pensing into each well 95 ll of nutrient broth and 5 ll of
the inocula. A hundred ll aliquot from the stock solu- 2.5.4. MIC agar dilution assay
tions of the essential oil and extracts initially prepared at MIC values of the fungal isolates were studied based
the concentration of 500 lg/ml was added into the first on the agar dilution method as described before
wells. Then, 100 ll from their serial dilutions was (Gulluce et al., 2003). The oil was added aseptically to
transferred into six consecutive wells. The last well sterile melted PDA medium containing Tween 20
containing 195 ll of nutrient broth without compound (Sigma 0.5%, v/v) at the appropriate volume to pro-
and 5 ll of the inocula on each strip was used as neg- duce the concentration range of 7.8–500 lg/ml. The
ative control. The final volume in each well was 200 ll. resulting PDA agar solutions were immediately poured
Maxipime (Bristol-Myers Squibb) at the concentration into Petri plates after vortexing. The plates were spot
range of 500–7.8 lg/ml was prepared in nutrient broth inoculated with 5 ll (104 spore/ml) of each fungi iso-
and used as standard drug for positive control. The plate late. Amphotericin B (Sigma A 4888) was used as a
was covered with a sterile plate sealer. Contents of each reference antifungal drug. The inoculated plates were
well were mixed on a plate shaker at 300 rpm for 20 s incubated at 27 and 37 C for 72 h for plant and
and then incubated at appropriate temperatures for 24 clinical fungi isolates, respectively. At the end of
h. Microbial growth was determined by absorbance at incubation period, the plates were evaluated for the
600 nm using the EL · 800 universal microplate reader presence or absence of growth. MIC values were
(Biotek Instrument Inc, Highland Park, Vermont, USA) determined as the lowest concentration of the essential
and confirmed by plating 5 ll samples from clear wells oil where absence of growth was recorded. Each test in
on nutrient agar medium. The extract tested in this this study was repeated, at least, twice.
 A. Sokmen et al. / Food Control 15 (2004) 627–634 631

Table 3
Antifungal and anticandidal activities of the essential oil and subfractions of the methanol extracts from Thymus spathulifolius against the yeast and
fungi isolates
Test yeast and fungi The oila Antibioticsb
DD MIC DD MIC (Amp B)
Yeast
Candida albicans-A117 12 250 – (NET) 31.25
Fungi
Alternaria alternata 36 125 – (NET) 15.62
Alternaria solani – – – (NET) NT
Aspergillus flavus 30 250 – (NET) 15.62
Aspergillus variecolor 21 250 – (NET) 62.50
Fusarium acuminatum 26 125 – (NET) 62.50
Fusarium culmorum – – – (NET) NT
Fusarium tabacinum 9 125 – (NET) 62.50
Fusarium oxysporum 31 62.50 – (NET) 62.50
Fusarium solani – – – (NET) NT
Fusarium pedis – – – (NET) NT
Penicillum spp. 32 62.50 – (NET) 31.25
Rhizopus spp. 35 125 – (NET) 125
Rhizoctonia solani 35 250 – (NET) 31.25
Moniliania fructicola 24 125 – (NET) 15.62
Trichophyton rubrum 12 31.25 – (NET) 15.62
Trichophyton mentagrophytes 18 125 – (NET) 31.25
Microsporum canis 20 125 – (NET) NT
Sclorotinia sclerotiorum 24 125 – (NET) 62.50
Sclorotinia minor 29 125 – (NET) 125
a
DD ¼ Inhibition zone in diameter (mm) around the discs impregnated with 10 ll of essential oil; MIC ¼ Minimal Inhibitory concentrations (lg/
ml) of the essential oil.
b
DD ¼ Inhibition zone in diameter (mm) around reference standards antibiotic discs. NET ¼ Netilmicin (30 lg/disc) was used as positive reference
standards antibiotic discs (Oxoid); MIC ¼ Minimal Inhibitory concentrations (lg/ml) of standard antibiotic, AmpB ¼ Amphotericin B (lg/ml)
(Sigma).

2.6. Antioxidant activity 2.6.2. b-Carotene–linoleic acid assay

In this assay antioxidant capacity is determined by
2.6.1. DPPH assay measuring the inhibition of the volatile organic com-
The hydrogen atoms or electrons donation ability of pounds and the conjugated diene hydroperoxides arising
the corresponding extracts and some pure compounds from linoleic acid oxidation (Dapkevicius et al., 1998). A
was measured from the bleaching of purple coloured stock solution of b-carotene–linoleic acid mixture was
methanol solution of DPPH. This spectrophotometric prepared as following: 0.5 mg b-carotene was dissolved
assay uses the stable radical diphenylpicrylhydrazyl in 1 ml of chloroform (HPLC grade), 25 ll linoleic acid
(DPPH) as a reagent (Burits & Bucar, 2000; Cuendet, and 200 mg Tween 40 was added. Chloroform was
Hostettmann, & Potterat, 1997). Fifty microlitres of completely evaporated using a vacuum evaporator.
various concentrations of the extracts in methanol was Then 100 ml distilled water saturated with oxygen (30
added to 5 ml of a 0.004% (w/v) methanol solution of min 100 ml min1 ) was added with vigorous shaking;
DPPH. After a 30 min incubation period at room tem- 2500 ll of this reaction mixture was dispersed to test
perature the absorbance was read against a blank at 517 tubes and 350 ll portions of the extracts prepared in
nm. Inhibition of free radical DPPH in percent (I%) was ethanol at 2 g l1 concentrations were added and the
calculated in following way: emulsion system was incubated for up to 48 h at room
temperature. The same procedure was repeated with the
I% ¼ ðAblank  Asample =Ablank Þ  100 positive control BHT and a blank. After this incubation
period, absorbance of the mixtures was measured at 490
where Ablank is the absorbance of the control reaction nm. Antioxidative capacities of the extracts were com-
(containing all reagents except the test compound), and pared with those of BHT at the same concentration and
Asample is the absorbance of the test compound. Extract blank consisting of only 350 ll ethanol.
concentration providing 50% inhibition (IC50 ) was cal-
culated using the graph by plotting inhibition percentage 2.7. Assay for total phenolics
against extract concentration. Synthetic antioxidant re-
agent butylated hyroxytoluene (BHT) was used as a po- Total phenolic constituents of polar and non-polar
sitive control and all tests were carried out in triplicate. subfractions of the methanol extract of T. spathulifolius
632 A. Sokmen et al. / Food Control 15 (2004) 627–634

was determined by employing the methods given in the amount of total phenolics in polar and non-polar sub-
literature (Chandler & Dodds, 1983; Slinkard & Single- fractions of the extract was estimated as 141 ± 0.90 lg/
ton, 1977) involving Folin-Ciocalteu reagent and gallic mg (14.1%, w/w) and 102 ± 0.55 lg/mg (10.2%, w/w),
acid as standard. An aliquot (0.1 ml) of extract solution respectively.
containing 1000 lg extract was added to a volumetric
flask, 46 ml distilled water and 1 ml Folin-Ciocalteu re-
agent were added and flask was shakenthoroughly. After 3.3. Antimicrobial activity
3 min, a 3 ml solution of Na2 CO3 (2%, w/v) was added
and the mixture was allowed to stand for 2 h with The in vitro antimicrobial activities of T. spathu-
intermittent shaking. Absorbance was measured at 760 lifolius essential oil and extracts against the micro-
nm. The same procedure was repeated with all standard organisms employed and their activity potentials were
gallic acid solutions (0–1000 lg per 0.1 ml) and a stan- qualitatively and quantitatively assessed by the pres-
dard curve was obtained using the equation given below: ence or absence of inhibition zones, zone diameters and
MIC values. According to the results given in Tables 2
Absorbance : 0:0012  Gallic acid ðlgÞ þ 0:0033: and 3, the essential oil of T. spathulifolius had great
potential of antimicrobial activities against all 25 bac-
teria, and 9 of 15 moulds and a yeast species tested.
3. Results and discussion Non-polar and polar subfractions of the methanol ex-
tract were also found to be effective against 18 and 13
3.1. Chemical composition of the essential oil out of 25 bacterial species examined, respectively (Ta-
ble 2). However, both of them failed to show anti-
Air-dried herbal parts of the plant were subjected to fungal or anticandidal activity. The maximal inhibition
hydrodistillation using a Clevenger apparatus and the zones and MIC values for bacterial strains, which were
yellow-coloured essential oil was obtained (yield 3.74% sensitive to the essential oil, and non-polar and polar
v/w). GC-MS analysis resulted in the identification of extracts of T. spathulifolius, were in the range of 7–32
twenty-eight compounds representing 99.2% of the oil. mm, and 7.81–500 ll/ml; 10–29 mm and 7.81–500 ll/
Thymol (36.5%), carvacrol (29.8%) and their bio- ml, and 9–27 mm and 31.25–500 ll/ml, respectively
synthetic precursors p-cymene (10.0%) and c-terpinene (Table 2). The maximal inhibition zones and MIC
(6.3%) were the main components comprising 82.6% of values of the yeast and fungi species sensitive to the
the oil (Table 1). The oil profile obtained from GC-MS essential oil of T. spathulifolius, were 12–36 mm and
analysis exhibits the presence of the major compounds, 31.25–250 ll/ml, respectively (Table 3). Based on these
e.g. thymol and carvacrol, almost in equal amounts. To results, it is possible to conclude that the essential oil
the best of our knowledge, the essential oil composition has a stronger activity and broader spectrum than
of T. spathulifolius has not been reported before and those of methanol extracts. As emphasised elsewhere,
therefore our results can be evaluated as the first report Gram-positive bacteria are more sensitive to plant oil
about the composition of the essential oil of this unique and extracts than Gram-negative bacteria (Cosentino
and endemic species. Compared to other endemic Thy- et al., 1999; Karaman et al., 2003). However, our re-
mus species collected from this flora, carvacrol content sults show that T. spathulifolius essential oil did not
of the oil presented here is lower than those of T. rev- possess any selective antimicrobial activity on the basis
olutus (43.13%) (Karaman et al., 2001), T. fallax (68.1%) of the cell wall differences of bacteria. By inhibiting the
(T€umen, Yildiz, Kirimer, K€ €o
urkcßu glu, & Baser, 1999), growth of all human and plant pathogenic and/or
but higher than those of T. pectinatus (3.7%) (Vardar- food spoilage bacteria, moulds and the yeast tested,
€ u et al., 2003) and T. pubescens var. cratericola
Unl€ T. spathulifolius essential oil exerted a broad anti-
€
(17.5%) (Baser, Ozek, K€urkcßu
€o
glu, T€umen, & Yildiz, microbial spectrum. The antimicrobial nature of the
1999). It would also be noteworthy to point out that the essential oil studied is apparently related to their high
composition of any plant essential oil studied is influ- phenolic contents, particularly carvacrol and thymol
enced by the presence of several factors, such as local, and this finding is in agreement with a previous report
climatic, seasonal and experimental conditions (Dafe- (Cosentino et al., 1999). This claim is further supported
rera, Ziogas, & Polissiou, 2000); thereby altering the by our findings; indicating high contents of thymol and
€ u et al., 2003).
biological activities studied (Vardar-Unl€ carvacrol; comprising 66.3% (v/v) of the oil (Table 1).

3.2. Amount of total phenolics 3.4. Antioxidant activity

Based on the absorbance values of the subfractions Free radical scavenging properties and the inhibition
reacted with Folin-Ciocalteu reagent and compared with effects on the lipid oxidation of various extracts of
the standard solutions of gallic acid equivalents, the T. spathulifolius are given in Table 4. Free radical
 A. Sokmen et al. / Food Control 15 (2004) 627–634 633

Table 4
Effects of T. spathulifolius essential oil, methanol extracts and positive control on the in vitro free radical (DPPH) scavenging and b-carotene/linoleic
acid assays
Sample DPPHa b-carotene/linoleic acidb
The oil 243.2 ± 7.20 92
Polar subfraction 16.15 ± 0.55 89
Non-polar subfraction 102.4 ± 1.50 82.4
BHT (positive control) 19.80 ± 0.50 96
a
IC50 values of DPPH assay (as lg/ml).
b
Given as percentage of % inhibition of the linoleic acid.

scavenging activity of the extracts is concentration bioactive properties and secrets could be lost forever
dependent and lower IC50 value reflects better protective without being tapped. Owing to its strong antibacterial
action. The polar subfraction of the methanol extract and excellent protective features exhibited in antioxidant
was able to reduce the stable free radical 2,2-diphenyl-1- activity tests, the essential oil and extracts from the
picrylhydrazyl (DPPH) to the yellow-colored diph- herbal parts of T. spathulifolius could be concluded as a
enylpicrylhydrazine with an IC50 of 16.2 ± 0.5 lg/ml, natural source that can be freely used in the food
exhibiting better activity than the synthetic antioxidant industry as a culinary herb, but, firstly, immediate and
agent BHT (19.8 ± 0.5 lg/ml). The aforesaid high total necessary measurements should be taken for the pro-
phenolic constituent of the polar subfraction (14.1%, w/ tection of this plant species. In conclusion, our study can
w, based on dry weight of the extract) as gallic acid be considered as the first report on the in vitro antimi-
equivalent also supports this activity suggesting that crobial and antioxidant properties of the essential oil
activity is mostly related to its water-soluble phenolic and methanol extracts prepared from T. spathulifolius.
compounds. The key role of phenolic compounds as We hope that our results introduce a unique natural
scavengers of free radicals is emphasised in several re- source which possesses strong antimicrobial and anti-
ports (Komali, Zheng, & Shetty, 1999; Moller, Madsen, oxidant substances.
Altonen, & Skibsted, 1999). The presence of polyphe-
nols, methoxylated flavonoids in particular, from Thy-
mus species was reported elsewhere (Adzet, Vila, &
Acknowledgements
Canigueral, 1988). Moreover, radical-scavenging activ-
ity is one of various mechanisms to contribute overall
Authors wish to thank to Mr. Yasar ZONGUR,
activity, thereby creating a synergistic effect.
technician, Department of Biology, Cumhuriyet Uni-
On the other hand, the inhibition values of linoleic
versity, Sivas-Turkey, for his help during the collection
acid oxidation were estimated as 92% and 89% in the
of plant material.
presence of the essential oil and polar subfraction of the
methanol extract, respectively (Table 4). Antioxidant
properties of thymol, carvacrol and c-terpinene were
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