South African Journal of Botany 149 (2022) 425
434

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South African Journal of Botany

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

Comprehensive study of chemical composition and biological activity of

Thymus pubescens Boiss. et Kotschy ex Celak.
Serkan Yigitkana,*, Mehmet Akdenizb, Ismail Yenerc, Zeki Sekerc, Mustafa Abdullah Yilmazc,
Mehmet Firatd, Deniz Evrim Kavake, Pelin Yilmaz Koseogluf, Abdulselam Ertasc,g, Ufuk Kolakf,1,
Ilkay Erdogan Orhanh
a
Department of Pharmacognosy, Faculty of Pharmacy, Dicle University, Diyarbakir 21280, Turkey
b
The Council of Forensic Medicine, Diyarbakir Group Chairmanship, Diyarbakir 21070, Turkey
c
Department of Analytical Chemistry, Faculty of Pharmacy, Dicle University, Diyarbakir 21280, Turkey
d
Department of Biology, Faculty of Education, Van Yuzuncu Yil University, Van 65080, Turkey
e
Science and Technology Center, Dicle University, Diyarbakir 21280, Turkey
f
Department of Analytical Chemistry, Istanbul University, Faculty of Pharmacy, Istanbul 34116, Turkey
g
Dicle University Cancer Research Center, Diyarbakir 21280, Turkey
h
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara 06330, Turkey

A R T I C L E I N F O A B S T R A C T

Article History: The use of Thymus species amongst the public and their importance in the scientific world is increasing day
Received 4 February 2022 by day. In addition to being consumed as tea and spice, Thymus species are used as folk medicine for respira-
Revised 27 April 2022 tory, digestive, skin, circulatory, genital, nervous, visual and urinary diseases. In this study, it was aimed to
Accepted 18 June 2022
examine the essential oil and ethanol extract of the root and aerial parts of Thymus pubescens Boiss. et Kot-
Available online 24 June 2022
schy ex Celak in the terms of their biological activity and chemical content. The phenolic content of the spe-
lu
Edited by: Prof U. Çakılcıog cies was determined by LC-MS/MS, while triterpenoid content, the chemical composition of the essential oil
Keywords:
as well as flavour was determined by GC
MS. In addition, their antioxidant and cytotoxic activities, as well
Thymus as acetyl- (AChE) and butyrylcholinesterase (BChE), urease, tyrosinase, elastase, collagenase, HMG-CoA
GC-MS reductase and angiotensin-converting enzyme (ACE) inhibitory activities were studied. Thymol (53.33%) was
LC-MS/MS determined as the major component in the essential oil, while b-fenchyl alcohol (43.87%) was determined as
Oleanolic acid the major component of the flavour. According to the LC-MS/MS and GC
MS results, it was found that espe-
Cytotoxicity cially the aerial parts of the species have a high amount of rosmarinic acid (3875.76 mg analyte/g extract),
Enzyme inhibition quinic acid (2392.55), naringenin (970.39), oleanolic (92785.96) and ursolic (63373.32) acids. The essential
Essential oil
oil of T. pubescens species was observed to show high activity in four antioxidant assays, e.g. inhibition of lipid
peroxidation, DPPH and ABTS radical scavenging activity as well as CUPRAC, while the ethanol extracts
showed moderate antioxidant activity. In enzyme inhibition assays, the aerial parts exerted marked BChE,
elastase, and collagenase inhibitory activities (92.43§1.28%, 42.59§0.56, and 48.61§0.39 at 100 mg/mL,
respectively). On the other hand, AChE, urease, tyrosinase, HMG-CoA reductase and ACE inhibitory activities
of all extracts were from low to moderate levels. In particular, the aerial parts of the species displayed a high
cytotoxic effect (vitality%: 6.82§0.01 at 200 mg/mL) in breast cancer (MCF-7) cell line. Due to its remarkable
antioxidant capacity, high content of rosmarinic, oleanolic, and ursolic acids and especially BChE, elastase,
and collagenase inhibitory activity, T. pubescens has the potential for using in food supplements, food preser-
vatives, cosmetics, and pharmaceutical industries.
© 2022 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction 215 species widely distributed in the world, especially in the Medi-
terranean region (El Yaagoubi et al. (2021)). Thymus species are rep-
The Lamiaceae family is a large family of dicotyledonous plants resented by 40 species and 42 taxa in Turkey, 16 of which are
comprising about 236 genera and 7200 species. Thymus L. genus is endemic and they are locally known as “kekik” in Anatolia as well as
amongst the most important genera of the Lamiaceae family, with “karabaş, kekik, ak kekik, sater, and nemamul otu” (Guner et al.,
2012; Selvi et al., 2013). Thymus species are sold as spice in many
* Corresponding author. countries. In Anatolian folk medicine, infusions prepared from the
E-mail address: [email protected] (S. Yigitkan).
1 f
aerial parts are consumed as antiseptic, stomachic, diuretic and

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0254-6299/© 2022 SAAB. Published by Elsevier B.V. All rights reserved.
S. Yigitkan, M. Akdeniz, I. Yener et al. South African Journal of Botany 149 (2022) 425
434

herbal tea against colds and it is used externally against excessive with Agilent 5977B model mass spectrometer (MS). Sample extrac-
vomiting and jaundice as an ointment (Tanker and Iliulu, 1981; Bay- tion for flavour analysis was done by solid phase microextraction
top, 1999; Selvi et al., 2013; Sargin, 2021; Kucukaydın et al., 2021). (SPME) method. Two grams of the sample taken directly from the dry
Thymus species contain many bioactive compounds. The main plant and it was transferred into a 20 mL headspace bottle and incu-
ones are terpenoids, flavonoids, and phenolic acids (Mahmoudi et al., bated at 40 °C for 15 min. The flavour compounds released during the
2008). Thymol, carvacrol, p-cymene and g -terpinene were identified incubation were adsorbed by the SPME fibre used and the adsorbed
as the main components of essential oils found in the genus Thymus components were sent to the GC
MS/FID device with the headspace
(Tohidi et al., 2017). In biological activity studies, T. pubescens Boiss. & sampling block. All chromatographic conditions were the same for
Kotschy ex Celak has been found to possess antioxidant, antimicro- essential oil and flavour analyses. GC
MS/FID analyses were per-
bial, antifungal, antibacterial, antileishmanial, anticancer, antispas- formed on nonpolar HP-5MS UI column (30 m £ 0.25 mm £ 0.25 mm
modic, and immunomodulatory effects (Mohseni and Rad, 2018). film) and helium was used as carrier gas. Identification of essential oil
Thymus species are known to be used in cardiovascular diseases, and flavour compositions was achieved by comparing the retention
hypertension, cholesterol, kidney stones, forgetfulness and various times and MS of the components. In addition, NIST and Wiley GC
MS
skin problems (Orch et al., 2020; El Yaagoubi et al. 2021). In this con- libraries were used to identify the components (Bakir et al., 2020;
text, there is no detailed study in the literature on T. pubescens so far, Fidan et al., 2021).
although it is widely used both for its pharmacological properties In order to analyse triterpene contents, the samples were derivat-
and as a spice and has a high economic value. In this study, especially ized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) contain-
the phenolic compounds of the root and aerial ethanol extracts of T. ing 1% trimethylchlorosilane (120 min at 70 °C) and examined by
pubescens were identified and quantified by LC-MS/MS. In addition, 7890A Model GC/FID with 5977B MS. Chromatographic separation
essential oil, flavour, and triterpene contents of the plant were clari- was performed with a nonpolar HP-5MS column
fied by GC
MS. Additionally, antioxidant properties of T. pubescens (30 m £ 0.25 mm £ 0.25 mm film). The GC oven was started at 200 °
were determined through b-carotene lipid peroxidation, 2,2- C, rapidly at 300 °C/min, and held constant for 15 min at this time
diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azinobis-(3-ethylbenzo- (300 °C). The temperature was then brought to 310 °C at a rate of 5 °
thiazoline-6-sulphonic acid) (ABTS) radical scavenging, and copper- C/min, which stabilized after 2 min. Fixed helium gas (0.8 mL/min)
reducing capacity (CUPRAC) assays. Besides, cytotoxic effect against was installed as the carrier gas. Injection and transfer line tempera-
HT-29 (colon cancer), MCF-7 (breast cancer), and PDF (healthy cell tures were set to 300 °C. Injections were made in splitless mode. The
lines), acetyl-(AChE) and butyryl-cholinesterase (BChE), urease, injection volume was 2.0 mL. The electron ionization/mass spectrom-
tyrosinase, elastase, collagenase, HMG-CoA reductase, and ACE inhib- eter (EI/MS) was set to an ionization energy of 70 eV. The ion source
itory activity of the essential oil and extracts of T. pubescens were was set to 230 °C. MS data were acquired by setting the full scan
examined. mode and scan m/z to a density of 50
650 atomic mass units (amu)
(Bakır et al., 2020).
2. Material and method
2.4. Total flavonoid-phenolic content and antioxidant activity assays
2.1. Plant material
The total phenolic and flavonoid contents of the extracts were cal-
Sample of Thymus pubescens Boiss. et Kotschy ex Celak was col-
culated as pyrocatechol and quercetin equivalents, respectively
lected from Van province by Mehmet Firat in July 2015 and identified
(Moreno et al., 2000; Slinkard and Singleton, 1977). The following
according to Flora of Turkey (Davis, 1970). The herbarium sample
equations were used for the total phenolic and flavonoid contents of
was stored in the herbarium of Van Yuzuncu Yil University, Faculty of
the prepared extracts, respectively.
Science (VANF) under code of M. Firat 32568. The current name of

the plant is written according to the International Plant Names Index Absorbance ¼ 0:0487 þ 0:0425 pyrocatecholðmgÞ r 2 ¼ 0:9950
and The Plant List.


Absorbance ¼ 0:0354 þ 0:0290 quercetin ðmgÞ r 2 ¼ 0:9938
2.2. Extraction and LC-MS/MS analyses
b-Carotene lipid peroxidation, ABTS cation and DPPH free radical
scavenging activity, and copper (II) ion reducing antioxidant capacity
The plant samples used were dried in the shade prior to extraction
(CUPRAC) assays were used to determine the antioxidant properties
process. While preparing the extracts used for biological activity and
of the samples (Miller, 1971; Re et, al., 1999; Blois, 1958; Apak et al.,
chemical content analysis, the root and aerial parts of the species
2004). In the mentioned four antioxidant test methods, a-tocopherol
were macerated with ethanol (3 £ 8 h). Each sample (5 g) was
and butylated hydroxytoluene (BHT) were used as the references.
extracted with 99% ethanol (1/5, w/v). Crude extracts were obtained
IC50 Calculations were made using samples with concentrations of
after filtration and solvent evaporation. Stock solutions were pre-
100, 50, 25, and 2.5 mg/mL.
pared from these crude extracts at concentrations of 4000 mg/mL,
diluted according to the studied method, and used for LC-MS/MS
analyses (Shimadzu 8040 model) and bioactivity assays. In this 2.5. Cytotoxic activity
method, thirty-seven phenolic- and flavonoid-derivative compounds
were determined in T. pubescens extracts using an LC-MS/MS method To determine the toxic and cytotoxic effects of the samples, the
previously developed and validated by our research group method of Mojarraba et al. (2013) was applied with minor modifica-
(Yilmaz et al., 2018). The LOD was between 0.05 and 25.8 mg/L and tions. The toxic effects of the samples were investigated against the
the LOQ was between 0.17 and 85.9 mg/L. The recoveries of phenolic PDF (healthy primary dermal fibroblast cell line) cell line and the
compounds varied between 96.9 and 106.2% (Yilmaz et al., 2018). cytotoxic effects against HT-29 (colon cancer cell line), and MCF-7
(breast cancer cell line). MTT assay was performed 48 h after treat-
2.3. GC conditions for triterpenoid, essential oil and flavour contents ment. Ten microliters of 5 mg/mL MTT solution was added to each
well and cells were incubated for 3 h at 37 °C with 5% CO2, 95% air
Components of essential oil samples (aerial parts dried in the and full humidity. After 3 h, the medium was removed and replaced
shade), obtained by hydrodistillation method using Clevenger appa- with 100 mL of DMSO. Plates were placed on a shaker for 15 min at
ratus, were identified using Agilent 7890A Model GC/FID combined room temperature and the optical density (OD) of the wells was
426
S. Yigitkan, M. Akdeniz, I. Yener et al. South African Journal of Botany 149 (2022) 425
434

determined at 540 nm using a plate reader (Multi Scan Go, Themo). 2.10. ACE inhibitory activity
Ethanol, which was used as the extraction solvent, was used in the
same volume as the control sample (Yener et al., 2018). The method developed by Kwon et al. (2006) was used with
minor modifications. Fifty microliters of sample solution was incu-
2.6. Anticholinesterase activity bated for 10 min at 25 °C with 200 mL of NaCl-borate buffer solution
(0.3 M NaCl, pH=8.3) containing 2 mU of ACE. After incubation, 100
The spectrophotometric method based on the inhibition of AChE mL of 5.0 mM substrate (hippuryl-histidyl-leucine) was added to the
and BChE developed by Ellman et al. (1961) was used to determine reaction solution and the solution was incubated at 37 °C for 1 h.
the anticholinesterase activity. Galantamine was used as the refer- Blank sample (containing buffer solution instead of enzyme and sub-
ence in this test method. strate), control (containing distilled water instead of sample extract)
and blank (containing sample extract and buffer instead of enzyme)
2.7. Anti-urease activity were also analysed. The reaction was terminated by adding 150 mL of
0.5 N HCL and the determination of the resulting hippuric acid was
The method developed by Hina et al. (2015) was used to deter- performed by high-performance liquid chromatography with an
mine urease inhibition potential of the samples. Thiourea was used ultraviolet detector (HPLC-UV) at 228 nm. Lisinopril was used as the
as the reference for the anti-urease activity assay. reference. ACE inhibition was calculated using the peak areas with
the following equation.


2.8. Anti-ageing activity Inhibition % ¼ Areacontrol

Areasample

Areasample blank

Tyrosinase, elastase, and collagenase inhibitory activity assays =ðAreacontrol

Areablank Þ  100
were used to determine the anti-ageing potential of the samples. The
method developed by Hearing and Jimenez (1987) for tyrosinase
3. Results and discussion
inhibitory activity was applied with minor modifications. Briefly, 150
mL of phosphate buffer (pH=8), 10 mL samples, and 20 mL enzyme 3.1. The results of LC-MS/MS
solutions (28 nM) were added to all wells and incubated at 37 °C for
10 min. Then, 20 mL of L-DOPA (0.5 mM) was added and incubated
In the current study, quantitative results of phenolic compounds
again for 10 min at 37 °C, and absorbence values were measured at
and their main and fragment ions of the root and aerial parts ethanol
475 nm. Kojic acid was used as the reference.
extracts of T. pubescens were determined using the LC-MS/MS
Elastase inhibitory activity was applied with minor modifications
method (Table 1). The analysis results exhibited that the aerial
using the method developed by Kraunsoe et al. (1996). Ten microli-
extract was very rich in rosmarinic acid (3875.76 mg analyte/g
ters of sample and 20 mL of elastase solution were added to 40 mL
extract). In addition, the aerial and root parts were found to have
(0.1 M Tris-Cl, pH=8) buffer solution and incubated for 10 min at 37 °
moderate levels in term of quinic acid (2392.55 and 657.92 mg ana-
C. Then, 30 mL of 1.015 mM substrate (N-succinyl-(Ala)
3-nitroani-
lyte/g extract, respectively).
lide) solution prepared with buffer solution (0.1 M Tris-Cl, pH=8) was
A literature survey showed that the aerial parts of plants were
added and incubated at 37 °C for 20 min. Then absorbence values
found to be more rich in phenolic compounds than those of the roots
were measured at 410 nm. Oleanolic acid was used as the reference.
(Ali et al., 2014; Akdeniz et al., 2021). In the study conducted in 2021
Collagenase inhibitory activity was performed with minor modifi-
on T. pubescens species, rosmarinic acid (70.4 mg/100 g), salvianolic
cations using the protocol developed by Thring et al. (2009). Twenty
acid (19.3 mg/100 g), and ferrulic acid (16.3 mg/100 g) were deter-
microliters of the sample solution prepared in DMSO, 10 mL of collage-
mined as the major compounds (Sarfaraz et al., 2021). In studies con-
nase solution (0.8 U/mL), and 50 mL of phosphate buffer (pH=7.5)
ducted on several Thymus species, i.e. T. glabrescens Willd., T.
were added to all wells and incubated at 25 °C for 15 min. Then, 20 mL
pannonicus All., T. praecox Opiz, T. pulegioides L., and T. serpyllum L.,
of substrate solution (N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala) was
the amount of rosmarinic acid, which was also determined as the
added and incubated at 25 °C for 20 min and absorbence values were
major compound, was found to be between 83.49 and 1436 mg/g dry
measured at 340 nm. Epicatechin gallate was used as the reference.
weight. Looking at previous studies, it was determined that rosmar-
inic acid was the major compound in Thymus species, similar to the
2.9. HMG-CoA reductase inhibitory activity 
present study (Boros et al., 2010; Cebovi c et al., 2018; Khouya et al.,
2019; Sarfaraz et al., 2021).
The enzyme inhibition method developed by Wang et al. (2015)
was applied by making minor changes. In the enzyme inhibition
3.2. Triterpenoid, essential oil and flavour contents
experiment procedure, firstly, 10 mL of the samples to be tested is
added to all wells. Then, 10 mL of potassium phosphate buffer pre-
The essential oil and flavour contents of T. pubescens were deter-
pared to contain EDTA (pH=7), dithiothrethiol (10 mmol/L), and
mined by GC
MS/FID and GC
MS/HeadSpace (Table 2). According to
bovine serum albumin (0.1 g/L) solution were added. Then, 20 mL of
the GC
MS/FID results, 96.56% of the essential oil and 36 compo-
enzyme solution with a final concentration of 4 U/mL and 40 mL of
nents were determined, while 95.97% of the flavour and 24 compo-
HMG-CoA (200 mM) solution were added and incubated at 37 °C for
nents were determined. As shown in Table 2, the monoterpene
5 min. Finally, 20 mL of NADPH was added and measured at 340 nm
hydrocarbon content of the flavour (28.02%) was richer than that of
in an ELISA microplate reader (Eon Biotek). Atorvastatin was used as
the essential oil (16.42%). The major components in the essential oil
the reference drug and DMSO as control. The following equation was
are thymol (53.33%), 1,8 cineol (7.40%), o-cymene (7.09%), endo-bor-
used to calculate the enzyme inhibitions of AChE, BChE, urease, tyros-
neol (5.17%), g -terpinene (3.24%) and carvacrol (2.48%), in flavour
inase, elastase, collagenase, and HMG-CoA reductase.
 b-fenchyl alcohol (43.87%), b-myrcene (9.53%), o-cymene (7.30%)
Enzyme inhibition %Þ ¼ 100
ðOD test well=OD controlÞ  100 and caryophyllene (6.29%), thymol (5.92%), and carvacrol (5.87%)
were determined as the main components. When the content of the
Inhibition% values of the samples in all enzyme inhibition meth- essential oil is examined in detail, it is seen that it is richer than the
ods were calculated at a concentration of 100 mg/mL. In addition, the flavour in terms of the number of components. It can be concluded
same volume of ethanol was used in enzyme inhibition assays. that the essential oil and flavour of T. pubescens are similar in terms
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S. Yigitkan, M. Akdeniz, I. Yener et al. South African Journal of Botany 149 (2022) 425
434

Table 1
Identification and quantification of phenolic compounds of T. pubescens ethanol extracts by LC-MS/MS.

No Analytes Rta Mother ion (m/z)b Fragment ionsc Quantification (mg analyte/g extract)d

TP-Re TP-AP

1 Quinic acid 1.13 190.95 85.3
93.3 657.92 2392.55

2 Malic acid 1.23 133.00 115.2
71.3 93.84 597.09
3 Fumaric acid 1.48 115.00 71.4 33.97 47.84
5 Protocatechuic acid 4.93 152.95 108.3 149.67 69.69
7 Chlorogenic acid 7.13 353.15 191.2 N.D. 36.11
8 4-OH-benzoic acid 7.39 136.95 93.3
65.3 77.94 N.D.
9 Vanillic acid 8.57 166.90 152.3
108.3 411.44 N.D.
10 Caffeic acid 8.80 178.95 135.2
134.3 N.D. 94.73
11 Syringic acid 9.02 196.95 182.2
167.3 N.D. 165.24
12 Vanillin 10.87 151.00 136.3
92.2 96.78 14.38
13 Salicylic acid 11.16 136.95 93.3
65.3 16.24 61.45
14 p-Coumaric acid 11.53 162.95 119.3
93.3 50.77 85.05
15 Rutin 12.61 609.05 300.1
271.1 N.D. 44.75
16 Ferulic acid 12.62 192.95 178.3 272.94 75.68
18 Hesperidin 12.67 610.90 303.1
465.1 54.70 143.43
19 Isoquercitrin 13.42 463.00 300.1
271.1 N.D. 72.95
20 Rosmarinic acid 14.54 359.00 161.2
197.2 N.D. 3875.76
23 Rhoifolin 16.11 577.05 269.2
211.1 9.40 313.07
25 Cosmosiin 16.59 431.00 268.2
239.2 N.D. 154.79
33 Naringenin 30.68 270.95 151.2
119.3 8.63 970.39
34 Apigenin 31.43 268.95 117.3
151.2 N.D. 112.10
a
Numbers on the far left raw indicate the standard phytochemical compounds. Undetected components: 4: Gallic acid, 6: Pyrocatechol, 17: Sinapinic acid, 21: Nicotiflorin, 22: o-
Coumaric acid, 24: Quercitrin, 26: Coumarin, 27: Myricetin, 28: Fisetin, 29: Cinnamic acid, 30: Liquiritigenin, 31: Quercetin, 32: Luteolin, 35: Hesperetin, 36: Kaempferol, 37:
Chrysin.
f
N.D: not detected.
a
Rt: Retention time.
b
Mother ion (m/z): Molecular ions of the standard compounds (m/z ratio).
c
Fragment ions: MRM fragments for the related molecular ions.
d
Values in mg/g (w/w) of plant ethanol extract.
e
The abbreviations for ethanol extracts (root, aerial part respectively) of the T. pubescens expressed as TP-R and TP-AR, respectively.

Table 2 Table 2 (Continued)

Chemical compositions of the essential oil and aroma of aerial parts of T. pubescens.
No RIa Constituentsb TP-Ec TP-Ac Identification
No RIa Constituentsb TP-Ec TP-Ac Identification Methods
Methods
36 1588 Spathulenol 0.56
Co-GC, MS, RI
1 928 3-Thujene 0.40 0.19 Co-GC, MS, RI 37 1595 Caryophyllene oxide 0.82
Co-GC, MS, RI
2 935 a-Pinene 1.47 2.32 Co-GC, MS, RI 38 1625 Epicubenol 0.26
Co-GC, MS, RI
3 951 Camphene 0.73 0.60 Co-GC, MS, RI 39 1650 tau-Cadinol 0.19
Co-GC, MS, RI
4 976 Sabinene 0.23 1.10 Co-GC, MS, RI 40 1664 a-Cadinol 0.21
Co-GC, MS, RI
5 980 b-Pinene 0.41 0.27 Co-GC, MS, RI Total identified (%) 96.56 95.97
6 985 3-Octanone 0.28
Co-GC, MS, RI Monoterpenes hydrocarbons 16.42 28.02
7 991 b-Myrcene 0.83 9.53 Co-GC, MS, RI Oxygenated monoterpenes 73.42 61.54
8 1019 a-Terpinene 0.55 0.56 Co-GC, MS, RI Sesquiterpenes hydrocarbons 4.68 6.41
9 1027 o-Cymene 7.09 7.30 Co-GC, MS, RI Oxygenated sesquiterpene 2.04

10 1031 D-Limonene 0.68 3.26 Co-GC, MS, RI
The essential oil and aroma of T. pubescens are expressed as TP-E and TP-A respectively.
11 1034 1.8 Cineole 7.40 2.94 Co-GC, MS, RI Co-GC
Co-injection with authentic compounds.
12 1047 cis-b-Ocimene 0.79 0.19 Co-GC, MS, RI RI
Retention Index literature comparison.
13 1060 g -Terpinene 3.24 2.48 Co-GC, MS, RI a
Kovats index on HP
5MS fused silica column.
14 1069 Sabinene hydrate 0.51
Co-GC, MS, RI b
A nonpolar Agilent HP-5MS fused silica column.
15 1091 Terpinolen
0.22 Co-GC, MS, RI c
Percentage concentration.
16 1101 Linalool
0.39 Co-GC, MS, RI
17 1149 Camphor
0.38 Co-GC, MS, RI
18 1170 endo-Borneol 5.17 0.95 Co-GC, MS, RI
19 1181 4-Terpineol 0.81 0.49 Co-GC, MS, RI
20 1195 b-Fenchyl alcohol 1.27 43.87 Co-GC, MS, RI of some major components e.g. o-cymene, g -terpinene, but differ in
21 1237 Thymol methyl ether 1.09 0.26 Co-GC, MS, RI terms of several major components e.g. b-fenchyl alcohol and thy-
22 1246 Carvacrol methyl ether 1.08
Co-GC, MS, RI
mol.
23 1252 Thymoquinon
0.47 Co-GC, MS, RI
24 1297 Thymol 53.33 5.92 Co-GC, MS, RI Literature survey showed similar studies on the chemical compo-
25 1303 Carvacrol 2.48 5.87 Co-GC, MS, RI sition of the essential oil of T. pubescens. The previous studies pointed
26 1393 b-Bourbonene 0.08
Co-GC, MS, RI out that the major components of the essential oil were in parallel
27 1429 Caryophyllene 2.52 6.29 Co-GC, MS, RI with the presented data, but the amount of thymol (53.33%), one of
28 1448 Aromandendrene 0.12
Co-GC, MS, RI
29 1463 a-Humulene 0.10 0.12 Co-GC, MS, RI
the major compounds, was found to be much higher than that of the
30 1484 g -Muurolene 0.15
Co-GC, MS, RI previous studies (Morteza-Semnani et al., 2006; Sefidkon et al.,
31 1490 Germacrene D 0.12
Co-GC, MS, RI 2002). Considering the studies on other species, the major compo-
32 1504 Varidiflorene 0.19
Co-GC, MS, RI nents for T. alsarensis Ronniger were linalool, carvacrol, and thymol,
33 1514 b-Bisabolene 0.80
Co-GC, MS, RI s;
while linalyl acetate, linalool, and a-terpineol for T. balcanus Borba
34 1523 g -Cadinene 0.16
Co-GC, MS, RI
35 1531 d-Cadinene 0.44
Co-GC, MS, RI carvacrol, p-cymene, g -terpinene for T. capitatus L., and T. vulgaris L.
as thymol, p-cymene, g -terpinene were the main ones. As can be
(continued) understood from these studies, it is obvious that the major
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S. Yigitkan, M. Akdeniz, I. Yener et al. South African Journal of Botany 149 (2022) 425
434

Table 3
Triterpenoid contents of T. pubescens ethanol extracts by GC
MS.

Compounds Rta Molecular ion-m/z RSDd% Three major fragment ions m/z TP-Rc TP-AP
(relative intensity%) (m/z)b (relative intensity%)

a-amyrin-TMS 17.99 498 (2.5) 0.025 218 (100) 203 (16.6) 189 (18.3) 419.565 NDe
Moronic acid-TMS 20.71 527 (21.1) 0.029 189 (100) 203 (40.3) 409 (24.3) ND ND
Oleanonic acid-TMS 20.96 527 (12.3) 0.023 203 (100) 408 (64.5) 189 (52.6) ND ND
Oleanolic acid-2TMS 21.55 601 (2.3) 0.026 203 (100) 189 (31.5) 320 (28.6) 14735.70 92785.96
Betulinic acid-2TMS 21.90 601 (4.9) 0.019 189 (100) 203 (34.5) 320 (21.8) 1509.79 ND
Ursolic acid-2TMS 22.55 601 (2.3) 0.015 203 (100) 189 (32.9) 320 (79.6) 12085.24 63373.32
Ursonic acid-TMSf 22.91 527 (9.5) 0.028 203 (100) 320 (60.4) 189 (24.9) ND ND
a
Rt: Retention time.
b
Mother ion(m/z): Molecular ions of the standard compounds (m/z ratio).
c
The abbreviations for ethanol extracts (root, aerial part) of the T. pubescens expressed as TP-R and TP-AR, respectively, mg analyte/g extract.
d
RSD: Relative standard deviation.
e
ND: Not detected,.
f
TMS: Tetrametilsilan.

components of essential oils of Thymus species vary according to the oleanolic and ursolic acids contents of T. pubescens aerial parts, it can
species, soil, and climatic conditions (Zeljkovic and Maksimovic, be commented to have potential to be used especially in the cosmetic
2015). industry.
When the triterpene content of the species is examined, it is seen
that the aerial part ethanol extract is quite rich in oleanolic and
ursolic acids (92785.96 and 63373.32 mg analyte/g extract, respec- 3.3. Total phenolic and flavonoid content
tively). In addition, a-amyrin, oleanolic, betulinic, and ursolic acids
were found in the root part of the plant (Table 3). When the results The results of the total phenolic and flavonoid contents of the root
were compared with the literature, it was determined that the and aerial ethanol extracts of the species were presented in Table 4.
amount of oleanolic and ursolic acids of T. pubescens was consider- Our findings showed that the phenolic content of the aerial ethanol
ably higher than the study conducted in 2016 (14.90 and 41.63 mg/ extract was higher than that of the root part (56.47§1.21 and 50.14§
100 g, respectively). Many researches showed that these two compo- 1.34 mg PEs/mg extract, respectively) in accordance with the LC-MS/
nents have anti-aging potential (Hong et al., 2012). Due to the high MS analyses. Likewise, it was determined that the flavonoid content

Table 4
Total phenolic-flavonoid content, antioxidant, and toxic-cytotoxic activities of T. pubescens extracts.

Samplesb Total phenolic contentc Total flavonoid contentd Antioxidant activity (mg/mL § S.D.a) Toxic-cytotoxic activity § S.D.
(mg PEs/mg § S.D.) (mg QEs/mg § S.D.)
IC50 vitality% at 200 mg/mL

Lipid peroxidation DPPH ABTS CUPRAC (A0.5) PDF HT-29 MCF-7

TP-R 50.14 § 1.34a 9.51 § 0.04 413.30 § 3.39 >1000 58.94 § 1.12 19.76 § 0.12 43.63 § 1.34 93.09 § 7.23 18.98 § 0.12
TP-AP 56.47 § 1.21 13.84 § 0.13 229.42 § 7.47 >1000 32.07 § 1.09 39.66 § 0.87 42.40 § 0.34 43.76 § 1.23 6.82 § 0.01
TP-E -e
49.60 § 1.78 547.56 § 9.54 4.88 § 0.08 15.86 § 0.03 67.98 § 1.35 60.88 § 1.56 12.10 § 0.21
BHTf

11.38 § 0.17 54.12 § 1.33 13.77 § 0.13 8.24 § 0.09



a-TOCf

15.23 § 0.22 16.43 § 0.24 9.88 § 0.34 18.27 § 0.67



a
Values expressed are means § S.D. of three parallel measurements and values were calculated according to negative control.
b
The abbreviations for ethanol extracts (root, aerial part and essential oil) of the T. pubescens expressed as TP-R, TP-AR and TP-E, respectively.
c
PEs: pyrocatechol equivalents (y = 0.0487x + 0.0425, r2=0.9950),.
d
QEs: quercetin equivalents (y = 0.0354x + 0.0290 r2=0.9938),.
e
No activity,.
f
Reference.

Table 5
Enzyme inhibitory activities of T. pubescens extracts.

Enzyme inhibition% § S.D.a at 100 mg/mL

Samplesb AChE BChE Urease Tyrosinase Elastase Collagenase HMG-CoA ACE

d
TP-R 3.38 § 0.01 29.95 § 0.67 NA 10.09 § 0.13 17.69 § 0.23 25.19 § 0.34 7.49 § 0.14 39.90 § 1.36
TP-AP NA 92.43 § 1.28 7.56 § 0.08 9.14 § 0.11 42.59 § 0.56 48.61 § 0.39 9.21 § 0.26 8.50 § 0.28
TP-E NA 34.93 § 1.04 31.15 § 1.60 NA 24.30 § 0.12 10.99 § 0.09 6.91 § 0.19 NA
Galantaminec 87.16 § 1.53 81.13 § 1.72






Thioureac

94.80 § 1.63





Kojic acidc


90.17 § 1.21




Oleanolic acidc



44.15 § 1.18



Epicatechin gallatec




85.79 § 1.88


Atorvastatinec





96.32 § 1.23

Lisinoprilc






99.80 § 1.18
a
Values expressed are means § S.D. of three parallel measurements and values were calculated according to negative control.
b
The abbreviations for ethanol extracts (root, aerial part and essential oil) of the T. pubescens expressed as TP-R, TP-AR and TP-E, respectively.
c
Reference.
d
Not active.

429
S. Yigitkan, M. Akdeniz, I. Yener et al. South African Journal of Botany 149 (2022) 425
434

Fig. 1. LC-MS/MS chromatograms A: TIC chromatogram of standard chemicals analysed by LC-MS/MS method. 1: Quinic acid, 2: Malic acid, 3: Fumaric acid, 4: Gallic acid, 5: Protoca-
techuic acid, 6: Pyrocatechol, 7: Chlorogenic acid,8: 4-OH Benzoic acid, 9: Vanillic acid, 10: Caffeic acid, 11: Syringic acid, 12: Vanillin, 13: Salicylic acid, 14: p-Coumaric acid, 15:
Rutin, 16: Ferulic acid, 17: Sinapinic acid, 18: Hesperidin, 19: Isoquercitrin, 20: Rosmarinic acid, 21: Nicotiflorin, 22: o-Coumaric acid, 23: Rhoifolin, 24: Quercitrin, 25: Cosmosiin,
26: Coumarin, 27: Myricetin, 28: Fisetin, 29: Cinnamic acid, 30: Liquiritigenin, 31: Quercetin, 32: Luteolin, 33: Naringenin, 34: Apigenin, 35: Hesperetin, 36: Kaempferol, 37: Chrysin,
B: LC-MS/MS chromatogram of the root part of T. pubescens ethanol extract, C: LC-MS/MS chromatogram of the aerial parts of T. pubescens ethanol extract.

of the aerial part was higher than that of the root part (13.84§0.13 activity of all samples was low, whereas the essential oil had higher
and 9.51§0.04 mg QEs/mg extract, respectively). antioxidant properties compared to those of the root and aerial etha-
nol extracts. In addition, the antioxidant capacity of the essential oil
3.4. Antioxidant and toxic/cytotoxic activities in the ABTS method (IC50: 4.88§0.08 mg/mL) was found to be stron-
ger than the references a-tocopherol and BHT.
Antioxidant activity of the ethanol extracts and essential oil of the Probing the literature, many studies on the antioxidant activity of
plant was determined by b-carotene bleaching assay, DPPH free and the genus Thymus can be found. For instance; in a study examining
ABTS cation radical scavenging activity along with CUPRAC method antioxidant activity of the essential oil of T. pubescens with DPPH
(Table 4). In general, we found that DPPH free radical scavenging method, it showed a low activity (10.24§0.35% inhibition) similar to
430
S. Yigitkan, M. Akdeniz, I. Yener et al. South African Journal of Botany 149 (2022) 425
434

Fig. 2. GC/FID-MS chromatograms, A: GC/FID-MS chromatogram of T. pubescens essential oil, B: GC/FID-MS chromatogram of T. pubescens flavour.

the DPPH radical scavenging activity results in the presented study at on breast and colon cancer lines. As reported in the previous studies,
a concentration of 100 mg/mL (Kucukbay et al., 2014). In other stud- the essential oils of several Thymus species and their components
ies with Thymus species, T. vulgaris L. (IC50: 4.80 mg/mL), T. serpyllum such as carvacrol, d-limonene, and thymol possessed cytotoxic effects
L. (IC50: 0.96 mg/mL), T. algeriensis Bioss & Reut (IC50: 1.64 mg/mL) both on cancer cell lines and in vivo studies (Deb et al., 2011;
and T. linearis Benth. (IC50: 42.9 mg/mL) showed high scavenging Jayakumar et al., 2012; Delgado-Adamez et al., 2017).
activity against DPPH radical (Hussain et al., 2013; Nikolic et al.,
2014; Salehi et al., 2019). In another study by Ertas et al. (2015), the 3.5. Enzyme inhibitory activities
methanol extract of T. nummularius M. Bieb. showed higher antioxi-
dant activity than those of BHT and a-tocopherol, which are used as When the results of anticholinesterase activities of the essential
references in all three of the DPPH, ABTS and lipid peroxidation oils and extracts of the studied Thymus species were examined
methods. It is seen that the high antioxidant capacities of various according to AChE and BChE inhibition methods, in general, it was
extracts of Thymus species, especially the aerial parts, are correlated determined that the aerial ethanol extract showed a high level of
with the amount of rosmarinic acid they contain. BChE inhibition (92.43§1.28%) (Table 5). When the urease inhibition
The toxic effects of the samples were determined by the MTT results were examined, the root part did not show any inhibition,
method on the healthy cell line (PDF), and the cytotoxic effects on while the essential oil and aerial part displayed a low level of inhibi-
the cancer cell line MCF-7 (breast cancer) and HT-29 (colon cancer) tion (31.15§1.60 and 7.56§0.08%, respectively). On the other hand,
(Table 4). In general, it was determined that the samples studied the essential oil did not show any inhibition against tyrosinase, while
herein showed a low level of toxic effect. Especially, the aerial part the ethanol extracts showed a low inhibition. It was also determined
ethanol extract showed high cytotoxicity (vitality% 43.76§1.23 that the aerial part had a high level of inhibition against elastase and
against HT-29 cell and 6.82§0.01 at 200 mg/mL against MCF-7 cell) collagenase enzymes (42.59§0.56 and 48.61§0.39%, respectively).
431
S. Yigitkan, M. Akdeniz, I. Yener et al. South African Journal of Botany 149 (2022) 425
434

Fig. 3. GC
MS chromatograms, A: TIC chromatogram of standard chemicals analysed by GC
MS method, B: GC
MS chromatogram of ethanol extract of T. pubescens root, C:
GC
MS chromatogram of the ethanol extract of T. pubescens aerial parts.

432
S. Yigitkan, M. Akdeniz, I. Yener et al. South African Journal of Botany 149 (2022) 425
434

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