Industrial Crops and Products 67 (2015) 336–345

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

A detailed study on the chemical and biological profiles of essential oil

and methanol extract of Thymus nummularius (Anzer tea):
Rosmarinic acid
Abdulselam Ertas a,∗ , Mehmet Boga b , Mustafa Abdullah Yilmaz c , Yeter Yesil d , Gulsen Tel e ,
Hamdi Temel c , Nesrin Hasimi f , Isil Gazioglu g , Mehmet Ozturk e , Pelin Ugurlu c
a
Department of Pharmacognosy, Faculty of Pharmacy, Dicle University, 21280 Diyarbakir, Turkey
b
Department of Pharmaceutical Technology, Faculty of Pharmacy, Dicle University, 21280 Diyarbakir, Turkey
c
Dicle University Science and Technology Research and Application Center (DUBTAM), Dicle University, 21280 Diyarbakir, Turkey
d
Department of Pharmaceutical Botany, Faculty of Pharmacy, Istanbul University, 34116 Istanbul, Turkey
e
Deparment of Chemistry, Faculty of Science, Muğla Sıtkı Koçman University, 48121 Muğla, Turkey
f
Department of Nutrition and Dietetics, School of Health, Batman University, 72060, Batman, Turkey
g
Deparment of Analytical Chemistry, Faculty of Pharmacy, Bezmialem Vakif University, Istanbul 34093, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to determine the chemical profile of Thymus nummularius by LC–MS/MS and
Received 30 October 2014 GC/MS. Additionally, the methanol extract, essential oil and some individual main compounds of Thy-
Received in revised form 28 January 2015 mus nummularius were tested for antioxidant, anticholinesterase and antimicrobial activities. Besides,
Accepted 30 January 2015
HPLC–FLD was used to determine total aflatoxin in the plant material. Among the twenty-seven com-
Available online 12 February 2015
pounds studied by LC–MS/MS, rosmarinic acid (131,898.9 ± 6463.0 ␮g/g dry-extract) was found to be the
most abundant compound in the methanol extract. On the other hand, thymol (60.38%) and terpinyl-
Keywords:
acetate (10.49%) were found to be the major compounds of the essential oil. Both the essential oil and
Thymus nummularius
Rosmarinic acid
the methanol extract of T. nummularius exhibited strong antioxidant activity in the four tested meth-
Antioxidant ods. Furthermore, the essential oil showed high inhibitory activity against acetyl-,butyryl-cholinesterase
Anticholinesterase enzymes and very strong antimicrobial activity against all tested microorganisms. Besides, T. nummula-
Antimicrobial rius can be used both as rosmarinic acid source and as food supplement due to its non-aflatoxin content
Essential oil and high antioxidant, anticholinesterase and antimicrobial activities.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
The genus Thymus L. is a member of Lamiaceae family and rep-
resented by 318 species in the world, 40 species in Turkey and 18
of them are endemic for Turkey (45%). Being a perrenial species,
Thymus nummularius M. Bieb. (synonym Thymus pseudopulegioides
Klokov & Des. -Shots.) is widely distributed in Caucasia (Jalas, 1982;
Yıldız, 2012). The plant is named as Anzer tea or Kekik (Ozbucak
et al., 2006) in Turkey and consumed as spices. In folk medicine, it is
used for its anthelmintic, palliative, lowering blood pressure effects.
It is also used to maintain mouth and teeth health (Baytop, 1994).
Furthermore, beekeepers believe that this species is the source of
∗ Corresponding author. Tel.: +90 412 248 8030/7536/505 628 3447.
E-mail addresses: abdulselamertas@hotmail.com, abdulselam.ertas@dicle.edu.tr
(A. Ertas).
famous Anzer Honey in East Black Sea Region of Turkey (Baytop,
1994).
In recent years, plant derivatives, especially essential oils, have
gained significant interest in academic society and food indus-
try (Ertas et al., 2014a; Costa et al., 2012; Albayrak and Aksoy,
2013; Pinto et al., 2014; Kasrati et al., 2014; Nikolic et al., 2014).
The screening of medicinal plants for efficacious compounds is
becoming increasingly important with the growing approval of
herbal medicine as an alternative form of health care. Essential oils
are concentrated hydrophobic liquids containing volatile aroma
compounds which are synthesized in several plant organs. These
molecules are a group of terpenoids, sesquiterpenes and possi-
bly diterpenes with different groups of aliphatic hydrocarbons,
alcohols, acids, acyclic esters, aldehydes and lactones (Tepe et al.,
2011). Essential oils derived from many plant species are known
to have antimicrobial and antioxidant activities, and attempts to
characterize their bioactive principles have increased in many
pharmaceutical and food-processing applications (Kucukbay et al.,

http://dx.doi.org/10.1016/j.indcrop.2015.01.064
0926-6690/© 2015 Elsevier B.V. All rights reserved.
 A. Ertas et al. / Industrial Crops and Products 67 (2015) 336–345
2014). Nevertheless, the use of phenolic compounds such as
flavonoids and phenolic acids that are plant based natural antiox-
idants, as well as preventive and therapeutic medicine is gaining
much appreciation. These kind of natural molecules are believed
to exhibit anticarcinogenic potential and present varied health-
promoting effects due to their antioxidant features (Costa et al.,
2012; Erdogan-Orhan et al., 2014).
Various types of spices behave as antimicrobial agents when
applied against different pathogenic bacteria and fungi in vitro.
Among the aromatic plants which belong to the family Lamiaceae,
essential oils and extracts of the genus Thymus are remarkable
for its high antimicrobial and antioxidant effects compared to the
essential oils and extracts of other plants (Kucukbay et al., 2014;
Tepe et al., 2011; Vallverdu-Queralt et al., 2014). The mentioned
biological properties are because of the chemical composition of
the essential oils and extracts which differ within the different
species of the genus Thymus and even within the samples of the
same species. Among the major compounds in the essential oil, thy-
mol and carvacrol were reported to have the highest antioxidant
activity (Kucukbay et al., 2014). Additionally, thymol and carvacrol
exhibit different biological activities. While thymol has an anti-
septic effect, carvacrol has antifungal characteristics (Tepe et al.,
2011). Nonvolatile antioxidant compound classes such as pheno-
lic acids and flavonoids were also found in the extracts of Thymus
species (Miron et al., 2011; Costa et al., 2012; Hyun et al., 2014;
Ali et al., 2014; Turumtay et al., 2014). Hence, essential oils and/or
non-volatile phytochemicals which exist in Thymus species can be
used as the natural preservative ingredients in the food industry.
Though there are numerous studies on the chemical com-
positions of the essential oils and extracts of different Thymus
species, studies on their biological activities are still limited. Thus,
after determining the essential oil composition of T. nummu-
larius with GC/MS (gas chromatograph/mass spectrometry), we
investigated both the aflatoxin content and associated antioxi-
dant, anticholinesterase and antimicrobial activities of the essential
oil and methanol extract. Furthermore, we analyzed twenty-
seven compounds in the methanol extract of T. nummularius with
LC–MS/MS for quantitative and qualitative purposes.
2. Materials and methods
2.1. Chemicals and instruments
Aflatoxin content of T. nummularius was determined by
HPLC–FLD (Shimadzu LC-20) and KOBRA-Cell derivatization
system. Also, chemical compositions of T. nummularius were inves-
tigated by using LC–MS/MS (Shimadzu, Kyoto, Japan) and GC–MS
instruments. A Shimadzu UV spectrophotometer and BioTek Power
Wave XS microplate reader (USA) were used for the activ-
ity assays. 2,2 ;-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS) (purity: 97.5%) and butylated hydrox-
ytoluene (BHT) (≥99%) were purchased from Merck (Germany);
(l)-malic acid (95–100%), quercetin (95%), protocatechuic acid
(97%), chrysin (97%), rutin (94%), hesperetin (95%), naringenin
(95%), rosmarinic acid (96%), vanillin (99%), p-coumaric acid (98%),
caffeic acid (98%), chlorogenic acid (95%), hyperoside (≥97%),
myricetin (≥96%), coumarin (≥99%), kaempferol (≥97%), formic
acid (≤100%), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (≥ 95%), ␤-
carotene (≥93%), linoleic acid (≥ 99%), tween 40, pyrocathecol
(≥99%), 5,5-dithiobis-(2-nitro benzoic acid) (DTNB) (≥98%), cop-
per (II) chloride dihydrate (CuCl2 .2H2 O) (≥99%), neocuproine
(2,9-dimethyl-1,10-phenanthroline) (≥98%), EDTA (Ethylenedi-
aminetetraacetic acid) (≥98%), acetylcholinesterase (AChE: from
electric eel) (Type-VI-S, EC 3.1.1.7, 425.84 U/mg), and butyryl-
cholinesterase (BChE: from horse serum) (EC 3.1.1.8, 11.4 U/mg)
337
were obtained from Sigma (Germany); quinic acid (98%), tr-
aconitic acid (98%), 4-hydroxybenzoic acid (≥99%), fisetin (≥98%),
␣-tocopherol (≥95.5%) and acetylthiocholine iodide (≥98%) were
from Aldrich (Germany); gallic acid (≥99%), tannic acid (puris), sal-
icylic acid (≥99%), and galanthamine hydrobromide (≥94%) were
from Sigma–Aldrich (Germany); Folin Ciocalteu Phenol reagent
was from Applichem (Germany); hesperidin (≥97%), luteolin
(≥97%), apigenin (≥99%), rhamnetin (≥99%) and butyrylthiocholine
iodide (≥99%) were from Fluka (Germany). Aflatoxin mix dissolved
in methanol was purchased from R-Biopharm as certified refer-
ence materials. Potassium bromide, HPLC grade acetonitril, HPLC
grade methanol, nitric acid and sodium chloride were purchased
from Merck, PBS (phosphate buffer saline) was purchased from
Sigma. Type 1 water was obtained Human water- purifying system.
Immunoaffinity columns were also purchased from R-Biopharm
(Darmstadt, Germany). All solvents were of analytical grade.
2.2. Plant material
The aerial parts of T. nummularius M. Bieb. (synonym Thymus
pseudopulegioides Klokov & Des. -Shots.) were identified and col-
lected from northern of Turkey (Trabzon) in July 2013 by Dr. Y.
Yesil. The specimen used to diagnose was stored in the Herbarium
of Istanbul University (ISTE 99821).
2.3. GC–MS and GC–FID conditions of essential oil
2.3.1. Preparation of essential oil
Essential oil was obtained using a Clevenger apparatus from the
dried aerial parts of T. nummularius (100 g) which were crumbled
into small pieces in distilled water (500 mL) for 3 h (Yield: 1.2%). The
obtained essential oil was dried over anhydrous Na2 SO4 and stored
at +4 ◦ C sufficient time. The essential oil was diluted by CH2 Cl2
(1:3 v/v) prior to GC–MS analysis.
2.3.2. Gas chromatography (GC)
A flame ionization detector (FID) and a VF-5MS fused silica cap-
illary non-polar column (30 m × 0.25 I.D., film thickness 0.25 ␮m)
were used for GC analyzes. The injector temperature and detector
temperature were adjusted to 250 and 270 ◦ C, respectively. Carrier
gas was helium at a flow rate of 1.4 mL/min. The sample size was
1.0 ␮L with a split ratio of 20:1. The initial oven temperature was
held at 60 ◦ C for 5 min, then increased up to 240 ◦ C with 4 ◦ C/min
increments and held at this temperature for 10 min. The percent-
age composition of the essential oil was determined by the Class
GC10 GC computer program.
2.3.3. Gas chromatography–Mass Spectrometry (GC–MS)
An ion trap MS spectrometer and a VF-5MS fused silica
non-polar capillary column (30 m × 0.25 mm I.D., film thickness
0.25 ␮m) were used for the GC–MS analyzes. Carrier gas was helium
at a flow rate of 1.4 mL/min. The oven temperature was held at 60 ◦ C
for 5 min, then increased up to 240 ◦ C with 4 ◦ C/min increments and
held at this temperature for 10 min. The injector and MS transfer
line temperatures were set at 220 and 290 ◦ C, respectively. The ion
source temperature was 200 ◦ C. The injection volume was 0.2 ␮L
with a split ratio of 1:20. EI–MS measurements were taken at 70 eV
ionization energy. Mass range was from m/z 28 to 650 amu. Scan
time was 0.5 s with 0.1 s inter scan delays. Identification of com-
ponents of the essential oil was based on GC retention indices and
computer matching with the Wiley, NIST-2005 and TRLIB library,
as well as by comparison of the fragmentation patterns of the mass
spectra with those reported in the literature (Adams, 1989) and,
whenever possible, by co-injection with authentic compounds.
338 A. Ertas et al. / Industrial Crops and Products 67 (2015) 336–345

Table 1
Analytical parameters of LC–MS/MS method.

Analyte no. Analytes RTa Equation r2 b RSD%c Linearity range (mg/L) LOD/LOQ (␮g/L)d Recovery (%) Ue

1 Quinic acid 3.36 f(x) = 25133 + 33.6x 0.9927 0.0388 250–10000 22.3/74.5 103.3 4.8
2 Malic acid 3.60 f(x) = −5674 + 93.6x 0.9975 0.1214 250–10000 19.2/64.1 101.4 5.3
3 tr-Aconitic acid 4.13 f(x) = −28416 + 79.3x 0.9933 0.3908 250–10000 15.6/51.9 102.8 4.9
4 Gallic acid 4.25 f(x) = 26417 + 358.1x 0.9901 0.4734 25–1000 4.8/15.9 102.3 5.1
5 Chlorogenic acid 5.29 f(x) = 26780 + 49.0x 0.9932 0.1882 250–10000 7.3/24.3 99.7 4.9
6 Protocatechuic acid 5.51 f(x) = 6197 + 36.9x 0.9991 0.5958 100–4000 25.8/85.9 100.2 5.1
7 Tannic acid 6.30 f(x) = 30233 + 90.3x 0.9955 0.9075 100–4000 10.2/34.2 97.8 5.1
8 tr-Caffeic acid 7.11 f(x) = 83958 + 1585.2x 0.9942 1.0080 25–1000 4.4/14.7 98.6 5.2
9 Vanillin 8.57 f(x) = −575 + 44.5x 0.9995 0.4094 250–10000 10.1/33.7 99.2 4.9
10 p-Coumaric acid 9.17 f(x) = 27064 + 73.5x 0.9909 1.1358 100–4000 15.2/50.8 98.4 5.1
11 Rosmarinic acid 9.19 f(x) = −1150 + 18.0x 0.9992 0.5220 250–10000 10.4/34.8 101.7 4.9
12 Rutin 9.67 f(x) = 3842 + 51.9x 0.9971 0.8146 250–10000 17.0/56.6 102.2 5.0
13 Hesperidin 9.69 f(x) = 105641 + 195.8x 0.9973 0.1363 250–10000 21.6/71.9 100.2 4.9
14 Hyperoside 9.96 f(x) = 827 + 1.0x 0.9549 0.2135 100–4000 12.4/41.4 98.5 4.9
15 4-OH Benzoic acid 11.38 f(x) = 54285 + 635.0x 0.9925 1.4013 25–1000 3.0/10.0 106.2 5.2
16 Salicylic acid 11.39 f(x) = 72571 + 915.2x 0.9904 0.6619 25–1000 4/13.3 106.2 5.0
17 Myricetin 11.42 f(x) = 5415 + 54.3x 0.9991 2.8247 100–4000 9.9/32.9 106.0 5.9
18 Fisetin 12.10 f(x) = 34409 + 331.9x 0.9988 2.4262 100–4000 10.7/35.6 96.9 5.5
19 Coumarin 12.18 f(x) = 34370 + 236.6x 0.9924 0.4203 100–4000 9.1/30.4 104.4 4.9
20 Quercetin 13.93 f(x) = 1693 + 206.1x 0.9995 4.3149 25–1000 2.0/6.8 98.9 7.1
21 Naringenin 14.15 f(x) = 39056 + 1100.6x 0.9956 2.0200 25–1000 2.6/8.8 97.0 5.5
22 Hesperetin 14.80 f(x) = 6545 + 160.3x 0.9961 1.0164 25–1000 3.3/11.0 102.4 5.3
23 Luteolin 14.84 f(x) = 3057 + 111.5x 0.9992 3.9487 25–1000 5.8/19.4 105.4 6.9
24 Kaempferol 14.85 f(x) = 571 + 21.0x 0.9917 0.5885 25–1000 2.0/6.6 99.1 5.2
25 Apigenin 16.73 f(x) = 18526 + 543.8x 0.9954 0.6782 25–1000 0.1/0.3 98.9 5.3
26 Rhamnetin 18.41 f(x) = 632 + 110.1x 0.9994 2.5678 25–1000 0.2/0.7 100.8 6.1
27 Chrysin 20.60 f(x) = 23532 + 698.8x 0.9965 1.5530 25–1000 0.05/0.17 102.2 5.3
a
RT: retention time.
b
r2 : coefficient of determination.
c
RSD: relative standard deviation.
d
LOD/LOQ (␮g/L): limit of detection/limit of quantification.
e
U (%): percent relative uncertainty at 95% confidence level (k = 2).

2.4. Identification and quantitation of phenolic compounds
2.4.1. Preparation of plant extract for biological activities and
LC–MS/MS
The air-dried and powdered plant materials (100 g) were
extracted three times with 300 mL of methanol for 24 h at room
temperature. The solvent was removed under vacuum at 30 ◦ C in a
rotary evaporator until dry methanol extracts were obtained (Yield:
15.6%). Dry filtrates were diluted to 250 mg/L and filtrated with
0.2 ␮m microfiber filter prior to LC–MS/MS analysis.
2.4.2. Instruments and chromatographic conditions for
LC–MS/MS
LC–MS/MS analyzes of the phenolic compounds were performed
by using a Nexera model Shimadzu UHPLC coupled to a tan-
dem MS instrument. The liquid chromatography was equipped
with LC-30AD binary pumps, DGU-20A3R degasser, CTO-10ASvp
column oven and SIL-30AC autosampler. The chromatographic sep-
aration was performed on a C18 reversed-phase Inertsil ODS-4
(150 mm × 4.6 mm, 3 ␮m) analytical column. The column temper-
ature was fixed at 40 ◦ C. The elution gradient consisted of mobile
phase A (water, 5 mM ammonium formate and 0.1% formic acid)
and mobile phase B (methanol, 5 mM ammonium formate and 0.1%
formic acid). The gradient program with the following proportions
of solvent B was applied t (min), B%: (0, 40), (20, 90), (23.99, 90), (24,
40), (29, 40). The solvent flow rate was maintained at 0.5 mL/min
and injection volume was settled as 4 ␮L.
2.4.3. MS instrumentation
MS detection was performed using Shimadzu LCMS 8040
model triple quadrupole mass spectrometer equipped with an ESI
source operating in both positive and negative ionization modes.
LC–MS/MS data were collected and processed by LabSolutions soft-
ware (Shimadzu, Kyoto, Japan). The multiple reaction monitoring
(MRM) mode was used to quantify the analyzes: the assay of
investigated compounds was performed following two or three
transitions per compound, the first one for quantitative purposes
and the second and/or the third one for confirmation.
2.4.4. Optimization of LC–MS/MS method
After different combinations of trials, in order to have rich
ionization and the separation of the molecules, gradient elution
was achieved using two solvents as (A) water (5 mM ammo-
nium formate and 0.1% formic acid) and (B) methanol (5 mM
ammonium formate and 0.1% formic acid). Among the most com-
monly used atmospheric pressure ionization sources such as ESI
(Electrospray ionization), APCI (atmospheric pressure chemical
ionization) and APPI (atmospheric pressure photoionization), ESI
source was chosen since the phenolic compounds were small and
relatively polar molecules. Furthermore, liquid chromatography
tandem mass spectrometry was decided to be used for the current
study due to its ion fragmentation stability (Ertas et al., 2014a,b).
The optimum ESI conditions were determined as interface tem-
perature; 350 ◦ C, DL temperature; 250 ◦ C, heat block temperature;
400 ◦ C, nebulizing gas flow (nitrogen); 3 L/min and drying gas flow
(nitrogen); 15 L/min.
2.4.5. Method validation parameters for LC–MS/MS
In this study, twenty-four phenolic compounds (flavonoids,
flavonoid glycosides, phenolic acids, phenolic aldehyde, coumarin)
and three non-phenolic organic acids which are widespread in
plant materials were qualified and quantified in T. nummularius.
Besides, among those compounds, chlorogenic, p-coumaric, caffeic
and rosmarinic acids, luteolin, kaempferol, quercetin and apigenin
are frequently detected in Thymus species and Lamiaceae family.
Rectilinear regression equations and the linearity ranges of the
studied standard compounds are given in Table 1 (Ertas et al.,
2014c). Correlation coefficients were found to be higher than 0.99.
 A. Ertas et al. / Industrial Crops and Products 67 (2015) 336–345 339

The limit of detection (LOD) and limit of quantitation (LOQ) of the
reported analytical method are shown in Table 1. For the stud-
ied compounds, LOD ranged between 0.05 and 25.8 ␮g/L and LOQ
ranged between 0.17 and 85.9 ␮g/L (Table 1). Moreover, the recov-
eries of the phenolic compounds ranged from 96.9% to 106.2%.
2.4.6. Estimation of uncertainty
2.4.6.1. Identification of uncertainty sources. Ertas et al. (2014a)
report was used to evaluate and quantify the uncertainty sources
of the applied method (Ertas et al., 2014a; Goren et al., 2004).
Purity of reference standards (pur), sample weights (w), cali-
bration curves (cal), repeatability (rep), stock solutions (Css) and
recovery (rec) were the sources used to calculate the uncertainties.
To calculate the standard combined uncertainty the following Eq.
(1) was applied:
 ration was performed at 40 ◦ C with isocratic elution. The mobile
u(C) = u2 (cal)+u2 (pur)+u2 (Css)+u2 (w)+u2 (rep)+u2 (rec)
Calibration curve and the purity of standards were defined as
the main sources of uncertainty. In order to calculate expanded
uncertainties at 95% confidence level (k:2), standard combined
uncertainties were multiplied by two. Calculated uncertainties are
given in Table 1.
2.5. Aflatoxin content by HPLC
2.5.1. Preparation of plant extract for aflatoxin analysis with
HPLC–FLD
Twenty-five grams of ground sample was weighed into a suit-
able container and 300 mL of methanol (70%) was added. After
shaking vigorously for 30 min with blender (Waring, 8011S), the
extract was filtered through Whatman no. 1 filter. 10 mL of the
obtained filtrate was diluted with 50 mL of PBS and 60 mL of the
supernatant were passed through an immunoaffinity column. The
column was washed with 10 mL of distilled water following the
passage of samples. Finally, the column was dried with air and
mycotoxins were eluted with 1.5 mL of methanol and 1.5 mL of
water was added into the vial. 100 ␮L of this eluate was injected
to HPLC for aflatoxin analysis.
2.5.2. Safety precautions, equipment and chromatographic
conditions for aflatoxin
Aflatoxins are toxic substances. They were always manipulated
in solution, avoiding the formation of dust and aerosols. Nitrile
gloves were used for all procedures.
The instrument used was a Shimadzu LC-20 liquid chromato-
graphic system equipped with a fluorescence detector (LC-20A) and
controlled by Lab Solutions software. Separation was achieved on
Cronusil-S ODS2C18 column (25 cm × 0.46 cm; 5 ␮m). A post col-
umn electrochemical derivatization was used to enhance aflatoxins
responses using KOBRA-Cell, 100 mA. The injection volume was
100 ␮L and the flow rate was 1.00 mL/min. Chromatographic sepa-
(1) phase used was mixture of acetonitrile, methanol, water (2; 3;
6 v/v), 0.132 g potassium bromide and 0.03% nitric acid. The wave-
lengths of excitation and emissions were fixed at 365 and 435 nm.
The LOD was 0.630 ␮g/kg for total aflatoxin and the recoveries were
between 83 and 96%.
2.6. Determination of total phenolic and flavonoid contents
Phenolic and flavonoid contents expressed as pyrocatechol and
quercetin equivalents respectively, were determined as reported in
the literature (Slinkard and Singleton, 1977; Moreno et al., 2000).
The following equations were used to calculate total phenolic and
flavonoid contents of the extracts:
Absorbance = 0.0238 + 0.0209 pyrocatechol (␮g) (r2 = 0.9968)
Absorbance = −0.1605 + 0.1571 quercetin (␮g) (r2 = 0.9946)
2.7. Antioxidant activity
We used the ␤-carotene-linoleic acid test system, DPPH free
radical and ABTS cation radical scavenging activities and cupric

Table 2
Chemical composition of the essential oil of Thymus nummularius (TNE).

RIa Constituentsb %c Identification methods RIa Constituentsb %c Idendification methods

827 2-Hexenal tr Co–GC, MS, RI 1199 Dihydrocarvone 0.03 MS, RI

922 ␣-Thujene 0.09 Co–GC, MS, RI 1235 Thymol methyl eter 2.56 MS, RI
932 ␣-Pinene 0.38 Co–GC, MS, RI 1250 Cis-geraniol 0.34 MS, RI
975 ␤-Pinene 0.29 Co–GC, MS, RI 1252 Linalyl acetate 3.09 MS, RI
979 1-Octen-3-ol 0.26 Co–GC, MS, RI 1286 Thymol 60.38 Co–GC, MS, RI
991 3-Octanol 0.05 Co–GC, MS, RI 1295 Carvacrol 1.90 Co–GC, MS, RI
1001 ␣-Phellandrene 0.04 MS, RI 1350 Terpinyl acetate 10.49 MS, RI
1015 ␣-Terpinene 0.39 Co–GC, MS, RI 1365 Neryl acetate 0.22 MS, RI
1023 p-Cymene 1.66 Co–GC, MS, RI 1370 ␣-Copaene tr Co–GC, MS, RI
1025 Limonene 0.26 Co–GC, MS, RI 1379 Geranyl acetate 0.44 MS, RI
1026 ␤-Phellandrene tr MS, RI 1380 ␤-Bourbonene 0.15 MS, RI
1057 ␥-Terpinene 3.12 Co–GC, MS, RI 1391 ␦-Elemene 0.08 MS, RI
1072 1-Nonen-3-ol tr Co–GC, MS, RI 1418 ␤-Caryophyllene 3.65 Co–GC, MS, RI
1099 Linalool 1.97 MS, RI 1440 ␣-Guaiene 0.02 MS, RI
1132 1-Octanol-acetate 0.16 Co–GC, MS, RI 1444 Aromadendrene 0.32 Co–GC, MS, RI
1166 Borneol 0.26 Co–GC, MS, RI 1449 ␣-Himachalene 1.27 MS, RI
1170 cis-Verbenol 0.03 Co–GC, MS, RI 1450 ␣-Humulene 0.14 Co–GC, MS, RI
1177 Terpinene-4-ol 0.29 Co–GC, MS, RI 1475 ␥-Muurolene 0.47 MS, RI
1187 p-Cymene-8-ol 0.04 MS, RI 1524 ␦-Cadinene 0.69 MS, RI
1188 ␣-Terpineol 3.01 Co–GC, MS, RI 1571 Spathulenol 0.50 MS,RI
Total identified (%) 99.04
Monoterpenes 6.23
Monoterpenoids 85.21
Sesquiterpenes 6.79
Sesquiterpenoids 0.50
Others 0.31

Co–GC: co-injection with authentic compounds, RI: retention index literature comparison, tr: trace.
a
Kovats index on DB – 1 fused silica column.
b
A nonpolar phenomenex DB-5 fused silica column.
c
Percentage concentration.
340 A. Ertas et al. / Industrial Crops and Products 67 (2015) 336–345

Fig. 1. LC–MS/MS chromatograms of A: 250 ppbstandard mix, B: Thymus nummularius methanol extract. 1: quinic acid, 2: malic acid, 3: tr-aconitic acid, 4: gallic acid, 5:
chlorogenic acid, 6: protocatechuic acid, 7: tannic acid, 8: tr- caffeicacid, 9: vanillin, 10: p-coumaric acid, 11: rosmarinic acid, 12: rutin, 13: hesperidin, 14: hyperoside, 15:
4-OH benzoic acid, 16: salicylic acid, 17: myricetin, 18: fisetin, 19: coumarin, 20: quercetin, 21: naringenin, 22: hesperetin, 23: luteolin, 24: kaempferol, 25: apigenin, 26:
rhamnetin, 27: chrysin.

reducing antioxidant capacity (CUPRAC) methods to determine the 3. Results and discussion
antioxidant activity (Miller, 1971; Blois, 1958; Re et al., 1999; Apak
et al., 2004; Ertas et al., 2014a). 3.1. Essential oil composition by GC–MS

2.8. Anticholinesterase activity
A spectrophotometric method developed by Ellman et al.
(1961) was used to indicate the acetyl- and butyryl-cholinesterase
inhibitory activities.
2.9. Antimicrobial activity
The antimicrobial activities of the extracts against different
microorganisms were assessed according to inhibition zone diam-
eter and MIC value (NCCLS, 1997, 2009).
2.10. Statistical analysis
The total phenolic-flavonoid contents, antioxidant and
anticholinesterase activity assay results were shown as
means ± standard deviation. The results were evaluated using
an unpaired t-test and one way analysis of variance ANOVA. The
differences were regarded as statistically significant at p < 0.05.
Essential oil was obtained using a Clevenger apparatus by
hydrodistillation from the aerial parts of T. nummularius. The essen-
tial oil composition was determined by GC/FID and GC/MS analysis.
Forty compounds consisting up to 99.04% of the essential oil were
identified. The retention indices, percentage concentration and
identification methods are given in Table 2. The major compounds
of the oil were found to be thymol (60.38%), terpinyl acetate
(10.49%), ␥-terpinene (3.12%) and linalyl acetate (3.09%). Monoter-
penoids represented 85.21% of the oil and sesquiterpenes 6.79%,
with monoterpenes 6.23%, sesquiterpenoids 0.50% and others 0.31%
as well (Table 2).
There are numerous studies on essential oil composition of
Thymus species in the literature. A literature survey about Thy-
mus species of Turkey reveals that main contents are ␣-terpineol
(30.6%) and thymol (20.7%) for T. migricus, carvacrol (66.1%) and
p-cymene (7.1%) for T. fallax, cis-carveol (29.6%) and ␣-terpinol
(10.8%) for T. pubescens var. pubescens (Kucukbay et al., 2014),
geraniol (55.0–59.1%) and geranyl acetate (27.1–28.8%) for T.
kotschyanus var. eriophorus and T. kotschyanus var. kotschyanus,
 A. Ertas et al. / Industrial Crops and Products 67 (2015) 336–345 341

Table 3
Identification and quantification of compounds of methanol extract of Thymus nummularius (TNM) by LC–MS/MS.

Compound Parention(m/z)a MS2 (Collision energy)b Quantification(mg analyte/g extract)c

Quinic acid 191.0 85 (22), 93 (22) 40696.6 ± 1953.4

Malic acid 133.1 115 (14), 71 (17) 9044.2 ± 477.5
tr-Aconitic acid 172.9 85 (12). 129 (9) 660.1 ± 32.2
Gallic acid 169.1 125 (14), 79 (25) 51.3 ± 2.6
Chlorogenic acid 353.0 191 (17) 842.3 ± 41.3
Protocatechuic acid 153.0 109 (16), 108 (26) 123.0 ± 6.2
Tannic acid 183.0 124 (22), 78 (34) 113.7 ± 5.8
tr-Caffeic acid 179.0 135 (15), 134 (24), 89 (31) 512.0 ± 26.7
Vanillin 151.1 136 (17), 92 (21) N.D.d
p-Coumaric acid 163.0 119 (15), 93 (31) 41.8 ± 2.1
Rosmarinic acid 358.9 161 (17), 133 (42) 131,898.9 ± 6463.0
Rutin 609.1 300 (37), 271 (51), 301 (38) 50.1 ± 2.5
Hesperidin 611.1 303,465 D.e
Hyperoside 463.1 300,301 1437.0 ± 70.4
4-OH Benzoic acid 137.0 93,65 162.7 ± 8.4
Salicylic acid 137.0 93,65,75 142.8 ± 7.1
Myricetin 317.0 179,151,137 N.D.
Fisetin 285.0 135,121 N.D.
Coumarin 147.0 103,91,77 D.
Quercetin 300.9 179,151,121 28.9 ± 2.0
Naringenin 271.0 151,119,107 408.0 ± 22.4
Hesperetin 301.0 164,136,108 N.D.
Luteolin 285.0 175,151,133 772.6 ± 53.3
Kaempferol 285.0 217,133,151 773.3 ± 40.2
Apigenin 269.0 151,117 282.3 ± 15.0
Rhamnetin 315.0 165,121,300 N.D.
Chrysin 253.0 143,119,107 N.D.
a
Parent ion (m/z): molecular ions of the standard compounds (mass to charge ratio).
b
MS2 (CE): MRM fragments for the related molecular ions (CE refers to related collision energies of the fragment ions).
c
Values in ␮g/g (w/w) of plant methanol extract.
d
N.D.: not detected.
e
D.: peak observed, concentration is lower than the LOQ but higher than the LOD.

carvacrol (57.2%) and p-cymene (11.0%) for T. Kotschyanus var.
glabrascens (Kucukbay et al., 2013), carvacrol (30.25%) and thymol
(18.32%) for T. hyemalis, carvacrol (41.34%) and p-cymene (19.80%)
for T. boveii (Tepe et al., 2011), thymol (70.82%) and p-cymene
(9.52%) for T. cappadocicus (Albayrak and Aksoy, 2013). It can be
observed that, the results obtained for these organic essential oils
of Thymus species were similar to those present in the scientific
literature.
3.2. Quantitative analysis of phenolic compounds by LC–MS/MS
In the current study, the total phenolic and flavonoid contents
were parallel to the LC–MS/MS results. Also it was determined that
the TNM (T. nummularius methanol) extract had a very rich phe-
nolic content because of its high level of rosmarinic acid amount
(131,899.0 ± 6463.0 ␮g/g dry extract) (Fig. 1 and Table 3). Like-
wise, the low total flavonoid content of this extract was parallel
to the low amount of flavonoids tested by LC–MS/MS. Along with
high level rosmarinic acid content of TNM extract, this extract
contained significant amount of chlorogenic (842.3 ± 41.3 ␮g/g),
protocatechuic (123.0 ± 6.2 ␮g/g), tr-caffeic (512.0 ± 26.7 ␮g/g), 4-
OH benzoic (162.7 ± 8.4 ␮g/g) and salicylic acids (142.8 ± 7.1 ␮g/g)
(Fig. 1 and Table 3). If LC–MS/MS results were examined in
terms of the flavonoids, relatively high amounts of hypero-
side (1437.0 ± 70.4 ␮g/g), kaempferol (773.3 ± 40.2 ␮g/g), luteolin
(772.6 ± 53.3 ␮g/g), naringenin (408.0 ± 22.4 ␮g/g) and apigenin
(282.3 ± 15.0 ␮g/g) took attention. Also it was shown that the
TNM extract contained low levels of rutin (50.1 ± 2.5 ␮g/g) and
quercetin (28.9 ± 2.0 ␮g/g). If we looked at the extract in terms
of non-phenolic compounds, it contains high amounts of quinic
(40,696.6 ± 1953.4 ␮g/g) and malic (9044.2 ± 477.5 ␮g/g) acids
(Fig. 1 and Table 3).
In a previous study regarding T. serpyllum, it was reported that
the aqueous extract contained high amounts of rosmarinic acid
as well as syringic, vanilic, chlorogenic, p-coumaric, caffeic acids
and the ethanol extract contained high amounts of luteolin and
apigenin (Miron et al., 2011). Numerous studies in the literature
reported the high amount of rosmarinic acid in Thymus species.
To illustrate, in a study it was reported that the ethanol extract of
T. quinquecostatus contains 31.3 ± 3.1 mg/g rosmarinic acid and the
water/ethanol extract of T. lotocephalus comprises 12.8 ± 31.9 mg/g
dry basis rosmarinic acid (Hyun et al., 2014; Costa et al., 2012).
When compared to literature, it is obvious that the phenolic acid
and flavonoid contents of Thymus species examined in this study are
parallel to other Thymus species in literature. However, this study
is the first one which reports such a high amount of rosmarinic
acid in a Thymus species. In this context, it could be said that T.
nummularius is a new source for rosmarinic acid (Table 3).
3.3. Determination of total aflatoxin by HPLC–FLD
T. numularius was uncontaminated in the terms of aflatoxin con-
tent (Fig. 2). Most of the analyzed food can get contaminated with
aflatoxins; pre-harvest, post-harvest, during the drying process,
transportation and/or in storage. Since mycotoxins in food and feed
cannot be completely avoided and the contaminated products may
cause significant health problems. Food control is very important to
protect human and animal health. The findings of the current study
exhibit that quality control of the foods around the world should
be done regularly and effectively.
3.4. Total phenolic and flavonoid contents
The total phenolic and flavonoid amounts were deter-
mined by expressing as pyrocatechol and quercetin equivalents,
respectively (y = 0.0238 + 0.0209 pyrocatechol (␮g), r2 = 0.9968
and y = −0.1605 + 0.1571 quercetin (␮g), r2 = 0.9946). The total
phenolic and flavonoid content of the TNM extract was charac-
342 A. Ertas et al. / Industrial Crops and Products 67 (2015) 336–345

Fig. 2. Aflatoxin chromatogram of A: aflatoxin standards and B: Thymus nummularius.

terized as 313.28 ± 3.74 ␮g PEs/mg extract and 25.74 ± 0.32 ␮g 3.5. Antioxidant activity
QEs/mg extract, respectively. It was detected that the total
phenolic contents were higher than total flavonoid contents The antioxidant activity of the methanol (TNM) extract and
(Table 4). essential oil (TNE) of T. nummularius and its main compounds

Table 4
Antioxidant activity* ,total phenolic-flavonoid contents* and anticholinesterase activity* of Thymus nummularius methanol extract, its essential oil, its main componenets,
BHT, ␣-TOC and galantamine.

Samples Inhibition% Inhibition% Phenolic content Flavonoid Lipid IC50 (␮g/mL) ABTS cation radical
against AChE against BChE (␮g PEs/mg extract)b content peroxidation DPPH free radical
(␮g QEs/mg
extract)c

TNM 13.31 ± 0.21a 47.18 ± 1.54a 313.28 ± 3.74 25.74 ± 0.32 6.54 ± 0.02a 5.73 ± 0.21a 7.10 ± 0.09a
RA 17.70 ± 0.11b 57.38 ± 0.09b – – 12.12 ± 0.02b 1.21 ± 0.06b 1.70 ± 0.07b
TNE 60.05 ± 0.37c 65.23 ± 0.59c – – 24.82 ± 0.08c 198.86 ± 1.35c 8.08 ± 0.49c
Thymol 73.18 ± 0.43d 68.36 ± 0.19d – – 19.51 ± 0.12d 250.39 ± 2.51d 5.47 ± 0.04d
Galantaminea 89.09 ± 0.29e 81.46 ± 0.09e – – – – –
␣-TOCa – – – – 13.42 ± 0.27b 19.61 ± 0.09e 9.92 ± 0.17e
BHTa – – – – 9.95 ± 0.19e 47.09 ± 0.12f 10.90 ± 0.16f

RA: rosmarinic acid.

*
Values expressed are means ± SEM of three parallel measurements (p < 0.05).
a
Standard drug.
b
PEs, pyrocatechol equivalents (y = 0.0238 + 0.0209x, r2 = 0.9968).
c
QEs, quercetin equivalents (y = –0.1605 + 0.1571x, r2 = 0.9946).
 A. Ertas et al. / Industrial Crops and Products 67 (2015) 336–345 343

4.5

4.0 TNM
3.5
TNE

Absorbance
3.0

2.5 RA

2.0 Thymol
1.5
BHT
1.0

0.5 α-TOC

0.0
0 10μg/mL 25μg/mL 50μg/mL 100μg/mL

Concentration

Fig. 3. Cupric reducing antioxidant capacity of Thymus nummularius methanol extract, its essential oil, its main components, ␣-tocopheroland BHT. RA: rosmarinic acid.

were investigated by using ␤-carotene bleaching, DPPH free radical
scavenging, cupric reducing antioxidant capacity and ABTS cation
radical decolorisation assays. To our knowledge, there is no study
about T. nummularius regarding these antioxidant methods in liter-
ature. As shown in Table 4, the antioxidant activity of the methanol
extract, essential oil and the main compounds of T. nummularius
were compared with those of ␣-tocopherol and BHT.
3.5.1. ˇ -Carotene-linoleic acid assay
Antioxidants play an essential role in lipid peroxidation inhibi-
tion or protection against cellular damages caused by free radicals.
Lipid peroxidation comprises a series of free radical mediated chain
reactions and related to various types of biological damages (Dargel,
1992). As shown in Table 4, the TNM extract and TNE of T. num-
mularius exhibited very strong lipid peroxidation activity (IC50 :
6.54 ± 0.02 and 24.82 ± 0.08 ␮g/mL, respectively) in ␤-carotene
bleaching method. Especially, the methanol extract exhibited
higher activity than ␣-tocopherol (IC50 :13.42 ± 0.27 ␮g/mL) and
BHT (IC50 :9.95 ± 0.19 ␮g/mL), which were used as standards in
the ␤-carotene bleaching method. Also, the essential oil exhibited
higher activity (90.98% inhibition) than ␣-tocopherol and BHT at
100 ␮g/mL. As seen in Table 3, TNM extract contains very high
amount of rosmarinic acid (131,898.85 ± 6463.00 ␮g/g dry extract).
Because of high ␤-carotene bleaching activity of rosmarinic acid as
seen in Table 4, it could be said that the observed activity of TNM
extract originated from synergic effect of rosmarinic acid and other
phenolic compounds in TNM extract. On the other hand, we want
to point to ␤-carotene bleaching activity (IC50 :19.51 ± 0.12 ␮g/mL)
and high amount of thymol (60.38%) in connection with the TNE
activity.
3.5.2. DPPH free radical and ABTS cation radical scavenging
activity
To determine the antioxidant chromogens assays attributed to
•
the use of DPPH• and ABTS + radicals are amongst the most widely
used spectrophotometric methods and free radicals can directly
•
react with antioxidants. Also, DPPH• and ABTS + scavenging meth-
ods are commonly used to determine the antioxidant activities
of compounds as they are simple, fast, sensitive and repeatable
(Ozcelik et al., 2003). As shown in Table 4, the TNM extract showed
very strong activity (IC50 : 5.73 ± 0.21 ␮g/mL) in DPPH free rad-
ical scavenging method that is higher than ␣-tocopherol (IC50 :
19.61 ± 0.09 ␮g/mL) and BHT (IC50 :47.09 ± 0.12 ␮g/mL) used as
standard compounds in DPPH method. The TNE showed weak activ-
ity (IC50 : 198.86 ± 0.35 ␮g/mL) in DPPH test. It can be said that this
high activity of TNM extract originates from its main compound
rosmarinic acid (Tables 3 and 4). Also, the low activity of TNE could
be related to thymol which also showed low activity in this method.
As shown in Table 4, the TNM extract and TNE indicated very
strong activity (IC50 : 7.10 ± 0.09 and 8.08 ± 0.49 ␮g/mL, respec-
tively) in ABTS cation radical scavenging assay. In addition, the
TNM extract and TNE exhibited higher activity than ␣-tocopherol
(IC50 : 9.92 ± 0.17 ␮g/mL) and BHT (IC50 : 10.90 ± 0.16 ␮g/mL). It can
be considered that these high activities of TNM extract and TNE
originate from their main components rosmarinic acid and thymol
(Tables 2–4).

Table 5
Antimicrobial activity of Thymus nummularius methanol extract, its essential oil and its main components against human pathogenic microorganisms.

Gram negative Gram positive Yeast

E. coli ATCC 25922 P. aeruginosa ATCC 27853 S. aureus ATCC 25923 S. pyogenes ATCC 19615 C. albicans ATCC 10231

TNE DDa 30.0 ± 0.5 27.0 ± 0.3 25.0 ± 0.2 24.0 ± 0.4 23.0 ± 0.3
MIC 15.0 ± 0.2 16.0 ± 0.2 21.0 ± 0.1 25.0 ± 0.4 27.0 ± 0.1

TNM DDb – 8.0 ± 0.1 9.0 ± 0.5 8.0 ± 0.3 11.0 ± 0.2
MIC – >1000 >1000 >1000 250.0 ± 0.3

Thymol DDb 18.0 ± 0.2 17.0 ± 0.5 21.0 ± 0.1 15.0 ± 0.4 15.0 ± 0.5
MIC 225.0 ± 0.4 850.0 ± 0.2 200.0 ± 0.3 300.0 ± 0.2 250.0 ± 0.4

RA DDb – 9.0 ± 0.3 9.0 ± 0.3 10.0 ± 0.5 –

MIC – >1000 >1000 >1000 –

Standards DDc 20.0 ± 0.1 – 35.0 ± 0.2 19.0 ± 0.2 30.0 ± 0.3
MIC 7.8 ± 0.4 – 1.9 ± 0.3 7.8 ± 0.1 3.1 ± 0.2

–: No inhibition zone and/or MIC value measured. TNE: essential oil of T. nummularius. TNM: methanol extract of T. nummularius. RA: rosmarinic acid.
a
DD: inhibition zone in diameter (mm) around the disks (6 mm) impregnated with 10 ␮L of essential oil.
b
DD: diameter of inhibition zone of methanol extract, thymol and/or rosmarinic acid at 30 mg/mL concentration.
c
DD: inhibition zone in diameter (mm) of positive controls that are ampicillin for bacteria and fluconazole for yeast. MIC: minimal inhibitory concentrations as ␮g/mL.
344 A. Ertas et al. / Industrial Crops and Products 67 (2015) 336–345
3.5.3. Cupric reducing antioxidant capacity
Cuprac method which is based on the reduction of Cu2+ to Cu+
was used in this study. This method is also of low cost, fast, selective
and suitable for various antioxidants without regard to their chemi-
cal structure and hydrophilicity (Apak et al., 2004). The TNM extract
(2.02 absorbance) and TNE (1.97 absorbance) exhibited almost the
same reducing effect in the CUPRAC assay at 100 ␮g/mL. Besides,
the TNM extract and TNE showed higher activity than ␣-tocopherol
(1.69 absorbance) in CUPRAC assay at 100 ␮g/mL. Rosmarinic acid
and thymol which are the main components of TNM and TNE,
showed 3.85 and 2.43 absorbance in CUPRAC assay at 100 ␮g/mL,
respectively (Fig. 3).
3.6. Anticholinesterase activity
To our knowledge, there is no study about anticholinesterase
activity of T. nummularius in literature. As shown in Table 4, the anti-
cholinesterase activity (against acetyl- and butyryl-cholinesterase
enzymes) of T. nummularius and individual main compounds
of the methanol extract and essential oil were compared with
that of galantamine, used as a standard drug against Alzheimer’s
disease. TNM extract showed moderate activity against butyryl-
cholinesterase enzyme (47.18 ± 1.54% inhibition) and weak activity
against acetyl-cholinesterase enzyme (13.31 ± 0.21% inhibition) at
200 ␮g/mL. The TNE exhibited good activity against acetyl- and
butyryl-cholinesterase enzymes (60.05 ± 0.37% and 65.23 ± 0.59%
inhibition, respectively) at 200 ␮g/mL. It is worth to say that
this good activity of TNE against acetyl- and butryl-cholinesterase
enzymes (73.18 ± 0.43 and 68.36 ± 0.19% inhibition, respectively)
was originated from the activity of thymol.
3.7. Antimicrobial activity
The antimicrobial activity of TNE, TNM, thymol and rosmarinic
acid were assessed against two gram-positive (Staphylococcus
aureus, Streptococcus pyogenes), two gram-negative (Escherichia
coli, Pseudomonas aeruginosa) bacteria and a yeast (Candida
albicans) using the disk diffusion method, followed by the mea-
surement of minimal inhibitory concentrations (MIC) (Table 5). TNE
exhibited strong antimicrobial activity (inhibition zone > 20 mm)
against all tested microorganisms. The antimicrobial activity of TNE
against gram negative bacteria was higher than the antibiotic used
as positive controls. The activity against gram positive bacteria and
yeast were very close to positive controls. The results indicated
that the most susceptible microorganism was E. coli (30 mm inhibi-
tion zone diameter and 15 ␮g/mL MIC value). TNM exhibited weak
antimicrobial activity (inhibition zone < 12 mm). Thymol was mod-
erately active on all microorganisms. TNE was more active than
thymol and rosmarinic acid that are the main component of TNE
and TNM, respectively.
Many Thymus species, except from T. nummularius, have been
investigated in terms of antimicrobial activity. Hussain et al. (2013)
lately reported the antimicrobial activity of T. linearis and T. serpyl-
lum essential oils against a panel of human and plant pathogenic
and food-borne bacterial strains. Results of the report revealed that
both of the essential oils possess good antimicrobial activity. In
a report comparing the essential oils of three different species of
Thymus (T. pallescent, T. algeriensis, T. dreatensis) collected from dif-
ferent regions, essential oils exhibited different activities against
the group of bacteria tested (Hazzit et al., 2009). Ballester-Costa
et al. (2013) have been investigated the antibacterial proper-
ties of T. zygis, T. mastichina, T. capitatus and T. vulgaris essential
oils and reported that the essential oils possess antibacterial
activity.
4. Conclusion
The results presented in this study are the first information
on the chemical profiles and antioxidant, anticholinesterase and
antimicrobial activities of the essential oil and methanol extract of
T. nummularius collected from Trabzon–Turkey.
Present study also showed that the TNM extract and TNE have
very strong activity in ␤-carotene bleaching, DPPH free radical
scavenging, cupric reducing antioxidant capacity and ABTS cation
radical decolorization assays. The antioxidant capacities of TNM
extract and TNE were parallel to the antioxidant properties of ros-
marinic acid and thymol which were the main components of these
extracts (Tables 2–4). In conclusion, the results revealed the impor-
tance of T. nummularius as an antioxidant which is one of the
Thymus species used as thyme in the world. Thus, the species may
help to protect the people against lipid peroxidation and free radical
damage, and its extracts will probably be used for the development
of safe food products and additives.
The TNE also demonstrated good acetyl- and butyryl-
cholinesterase inhibitory activities. As the main compound of the
essential oil, thymol exhibited better inhibitory activity than TNE
(Table 4). Thymus species are known to be rich in thymol. Therefore,
consumption of thymol rich Thymus species may be useful as good
cholinesterase inhibitory agents.
Additionally, TNE has strong antimicrobial activity. On the other
hand, thymol which is the main component of TNE exhibited mod-
erate antimicrobial activity. The activity of TNE should be due to
the synergistic effect of components.
The results of LC–MS/MS analysis of TNM extract indicate that
it can be used as a new source of rosmarinic acid. This finding is the
most stunning result of this study.
As a consequence of this study, consumption of T. nummularius
may protect people against oxidative stress, pathogenic microor-
ganisms and amnesia without any side effect. However, further
studies, particularly in vivo tests, are needed to understand the
activity in biological systems.
Acknowledgments
The authors are thankful to Dicle University Science and Tech-
nology Research and Application Center (DUBTAM) for its support
in this study. We acknowledge the Dicle University for financial
support (Research University Grant DUBAP: 14-EZF-69).
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