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antioxidants
Article
Thymus zygis subsp. zygis an Endemic Portuguese
Plant: Phytochemical Profiling, Antioxidant,
Anti-Proliferative and Anti-Inflammatory Activities
Amélia M. Silva 1, 2, * , Carlos Martins-Gomes 2,3 , Eliana B. Souto 4,5 , Judith Schäfer 6,
João A. Santos 2,7 , Mirko Bunzel 6and Fernando M. Nunes 3, 8, *
1Department of Biology and Environment, School of Life Sciences and Environment, University of
Trás-os-Montes and Alto Douro (UTAD), 5001-801 Vila Real, Portugal
2Centre for Research and Technology of Agro-Environmental and Biological Sciences (CITAB), UTAD,
5001-801 Vila Real, Portugal; camgomes@utad.pt (C.M.-G.); jsantos@utad.pt (J.A.S.)
3Food and Wine Chemistry Lab., Chemistry Research Centre-Vila Real (CQ-VR), UTAD,
5001-801 Vila Real, Portugal
4Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências
da Saúde, 3000-548 Coimbra, Portugal; ebsouto@ff.uc.pt
5CEB–Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
6
Department of Food Chemistry and Phytochemistry, Institute of Applied Biosciences, Karlsruhe Institute of
Technology (KIT), Adenauerring 20a, Building 50.41, 76131 Karlsruhe, Germany;
judith.schaefer@kit.edu (J.S.); mirko.bunzel@kit.edu (M.B.)
7Department of Physics, School of Sciences and Technology, UTAD, 5001-801 Vila Real, Portugal
8Department of Chemistry, School of Life Sciences and Environment, UTAD, 5001-801 Vila Real, Portugal
*Correspondence: amsilva@utad.pt (A.M.S.); fnunes@utad.pt (F.M.N.);
Tel.: +351-259-350-921 (A.M.S.); +351-259-350-907 (F.M.N.)
Received: 27 April 2020; Accepted: 29 May 2020; Published: 3 June 2020
Abstract:
Thymus zygis subsp. zygis is an endemic Portuguese plant belonging to the Thymus zygis species.
Although T. zygis is commonly used as a condiment and as a medicinal herb, a detailed description of
the polyphenol composition of hydroethanolic (HE) and aqueous decoction (AD) extracts is not available.
In this work, we describe for the first time a detailed phenolic composition of Thymus zygis subsp. zygis HE
and AD extracts, together with their antioxidant, anti-proliferative and anti-inflammatory activities. Unlike
other Thymus species, T. zygis subsp. zygis extracts contain higher amounts of luteolin-(?)-O-hexoside.
However, the major phenolic compound is rosmarinic acid, and high amounts of salvianolic acids K
and I were also detected. T. zygis subsp. zygis extracts exhibited significant scavenging activity of ABTS
+
,
hydroxyl (
•
OH), and nitric oxide (NO) radicals. Regarding the anti-proliferative/cytotoxic effect, tested
against Caco-2 and HepG2 cells, the AD extract only slightly reduced cell viability at higher concentrations
(IC
50
>600
µ
g/mL, 48 h exposure), denoting very low toxicity, while the HE extract showed a high
anti-proliferative effect, especially at 48 h exposure (IC
50
of 85.01
±
15.10
µ
g/mL and 82.19
±
2.46
µ
g/mL,
for Caco-2 and HepG2, respectively). At non-cytotoxic concentrations, both extracts reduced the nitric
oxide (NO) release by lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (at 50
µ
g/mL, HE and AD
extracts inhibited NO release in ~89% and 48%, respectively). In conclusion, the results highlight the
non-toxic effect of aqueous extracts, both resembling the consumption of antioxidants in foodstuff or in
functional food. Furthermore, the HE extract of T. zygis subsp. zygis is a source of promising molecules with
antioxidant, anti-inflammatory and anticancer activities, highlighting its potential as a source of bioactive
ingredients for nutraceutical and pharmaceutical industries.
Keywords:
Thymus zygis subsp. zygis; phenolic profiling; aqueous decoction; hydroethanolic
extract; luteolin-O-hexoside; anti-proliferative activity; radical scavenging activity; antioxidant;
anti-inflammatory activity
Antioxidants 2020,9, 482; doi:10.3390/antiox9060482 www.mdpi.com/journal/antioxidants
Antioxidants 2020,9, 482 2 of 20
1. Introduction
The genus Thymus, belonging to the Lamiaceae family, includes ca. 350 species of perennial,
subshrubs, and aromatic herbs native to Europe and North Africa, with many of them being endemic
to the Mediterranean region [
1
–
3
]. Thymus plants are heliophylous, thus they grow well in a climate
with moderate to warm temperatures, in well-drained to dry soils (usually they grow on rocks, stones,
or sand), and in sunny places [
4
]. Besides these ecological preferences, some Thymus species are easily
cultivated, especially in calcareous light, dry, stony soils and heavy wet soils, but lose some aromatic
properties [4].
Thymus zygis Loefl. ex L. (Lamiaceae) grows in the countries around the Mediterranean
Sea and is widespread throughout Portugal and Spain [
5
,
6
]. Thymus zygis is commonly named
“erva-de-Santa-Maria”; “sal-da-terra”, “serp
ã
o-do-monte” (Portuguese), white-thyme, and others [
7
,
8
].
For this species, three subspecies are described, namely Thymus zygis subsp. zygis Loefl. ex L.;
Thymus zygis subsp. gracilis (Boiss.) R. Morales, and Thymus zygis subsp. sylvestris (Hoffmanns. & Link)
Coutinho [
1
,
9
]. In Portugal, only two of these subspecies are found, T. zygis subsp. zygis and T. zygis
subsp. sylvestris, which present differences in some botanical characteristics, chromosome number,
and ecology [
6
,
9
]. However, in Spain, it is possible to find T. zygis subsp. gracilis, instead of T. zygis
subsp. zygis [
5
,
10
]. In Portugal, T. zygis L. subsp. sylvestris is commonly found in the central regions,
and it is traditionally used in the preservation of food (e.g., olives), as a condiment (e.g., in cheese,
fish, meat, salads, sauces), as a digestive tonic, and in the treatment of colds and sore throat [
11
].
T. zygis subsp. gracilis (known as red thyme) essential oil is rich in thymol, and due to its relevance
in thyme essential oil quality, T. zygis has become the most commercialized thyme in Spain because
of its economic importance [
12
]. Indeed, T. zygis is amongst the five thyme species with the highest
commercial value, together with Thymus vulgaris L. (common thyme), Thymus capitatus (L.) Hoffmanns.
et Link (recently classified as Thymbra capitata (L.) Cav.), Thymus mastichina L., Thymus serpyllum L.,
mostly due to the essential oils, but T. vulgaris and T. zygis have also high economic values for culinary
and seasonings, mostly sold as dry herbs [1].
Although the essential oil composition of the different T. zygis subspecies has been thoroughly
described in the literature [
6
,
12
–
14
], together with the related bioactive properties [
11
,
13
,
15
], studies
concerning its polyphenol composition and bioactivity are scarcer. The methanolic and ethyl
ether extracts of T. zygis were however shown potent antioxidant activity resulting from a direct
correlation with their phenolic content [
16
,
17
]. As far as we known, only three studies report the
polyphenolic composition of T. zygis, with one of these being performed in T. zygis (subspecies
gracilis) hydrodestillation by-products aiming the valorization of this abundant waste [
18
]. In other
study, water extraction of T. zygis (subspecies not described) polyphenols, mimicking a decoction
preparation, revealed rosmarinic acid as the major phenolic compound, and showed antioxidant and
anti-bacterial activities towards Gram-positive Staphylococcus aureus and Staphylococcus epidermidis and
Gram-negative Escherichia coli,Salmonella typhimurium and Pseudomonas aeruginosa bacteria [
19
]. In the
third study, aqueous extracts of T. zygis subsp. gracilis, rich in caffeic and rosmarinic acids, revealed
moderate to high antioxidant activity, potent anti-inflammatory activity (in a mice model of croton
oil-induced ear edema), and strong anticoagulant activity [
20
]. Anti-inflammatory, antioxidant, and
anticoagulant effects were also described for non-characterized T. zygis aqueous extracts [
21
]. In a study
involving several medicinal plants, methanolic extracts of T. zygis (the three subspecies) showed potent
anti-bacterial activity against the Gram-positive Staphylococcus aureus and Bacillus cereus, which was
correlated with the high total phenolic content (quantified with Folin–Ciocalteau’s reagent) obtained
for these extracts [17].
Although few research articles refer to the T. zygis phenolic composition, none offered a
complete/exhaustive description. T. zygis, together with T. vulgaris, is mentioned by the European
Medicines Agency (EMA) through the Committee on Herbal Medicinal Products (HMPC), and
approved in several pharmaceutical preparation forms, in which water and ethanol are the main
recommended solvents [
22
,
23
]. Indeed, T. zygis products (extracts and/or essential oils) are used as an
Antioxidants 2020,9, 482 3 of 20
ingredient in several pharmaceutical and dietary supplement preparations, such as anti-cough syrups
(e.g., Sideri-Bronsid, from Sideri Laboratory, Belgium; Pertusinas
®
Forte, from VALENTIS, Lithuania),
anti-cough pastilles (e.g., Buttercup Bronchostop Cough Pastilles; Omega Pharma Ltd., London, UK),
expectorant and anti-cough syrups (e.g., hydraSense
®
Mucus & Phlegm Cough Syrup, from Bayer
AG, Leverkusen, Germany), mouth and throat sprays (e.g., LaDr
ô
me Propolis throat and mouth spray,
Ladrôme Laboratoire, Saillans, France), and others.
Another important field of T. zygis application is in the livestock and food industry. The inclusion
of T. zygis subsp. gracilis leaves in the diet of pregnant sheep was reported to positively affect the
sensorial characteristics, as well as the oxidative stability of cooked lamb meat [
24
]. Goats fed with
distilled and non-distilled T. zygis subsp. gracilis leaves showed an improvement in the sensory
(reduced lipid oxidation) and nutritional properties (increased content in protein, fat, dry matter,
and polyunsaturated fatty acids) of milk, as well as of cheese [
25
]. Rabaçal cheese (PDO, protected
designation of origin), produced in central Portugal where sheep and goat are fed freely in fields rich
in T. zygis, has a distinctive characteristic aroma and flavor, attributed to the thyme [
26
]. To the best of
our knowledge, there is no scientific report in which the presence of T. zygis bioactive compounds was
characterized/quantified in Rabaçal cheese.
As the phenolic composition of T. zygis, and especially that of T. zygis subsp. zygis, is not fully
known, due to its potential bioactivities and economic value, the main objective of this work was to
determine the polyphenol composition of T. zygis subsp. zygis hydroethanolic extracts, by applying an
exhaustive extraction procedure to access the whole extractable polyphenol composition, and aqueous
decoction extracts, mimicking the human consumption as herbal tea. Together with the phenolic profile
of T. zygis subsp. zygis extracts, this work also aims to characterize the extract’s anti-oxidant activity,
anti-proliferative/cytotoxic activity against Caco-2 and HepG2 cell lines, and the anti-inflammatory
activity, using the LPS-stimulated RAW 264.7 cell model.
2. Materials and Methods
2.1. Standards and Reagents
Methanol (HPLC or MS grade), ethanol, formic acid, acetic acid, hydrogen peroxide
(30% solution), trichloroacetic acid (TCA), Folin–Ciocalteau’s reagent, 2-deoxy-D-ribose, sodium
nitrite, sodium nitroprusside, potassium persulfate, sodium molybdate, aluminum chloride (III),
ethylenediaminetetraacetic acid (EDTA), ascorbic acid, sulfanilamide, N-(1-naphthyl)ethylenediamine
dihydrochloride, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS),
(
±
)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), thiobarbituric acid (TBA),
and standards of rosmarinic acid, catechin, luteolin, apigenin, and ursolic acid were purchased from
Sigma-Aldrich/Merck (Alg
é
s, Portugal). Caffeic acid was obtained from Extrasynthese
®
(Genay, France).
Oleanolic acid was obtained from Santa Cruz Biotechnology Inc. (Frilabo; Porto, Portugal). Dulbecco’s
Modified Eagle Medium (DMEM), sodium pyruvate, penicillin, streptomycin, versene, L-glutamine,
trypsin-EDTA, and foetal bovine serum (FBS) were obtained from Gibco (Alfagene, Invitrogen,
Portugal). Alamar Blue®was obtained from Invitrogen, Life-Technologies (Porto, Portugal).
2.2. Plant Material
T. zygis subs zygis (T. zygis) aerial parts (upper part of stems, their leaves and flowers) were grown
in organic farming conditions and harvested in April 2016 (beginning of flowering stage) in Mezio,
Viseu, Portugal; at 40
◦
58’47.4” N 7
◦
53’43.3” W and supplied by ERVITAL
®
(Plantas Arom
á
ticas e
Medicinais, Lda). A voucher specimen (T. zygis subsp. zygis: Voucher N. HVR21092) was deposited
in the botanical garden office at the University of Tr
á
s-os-Montes and Alto Douro (UTAD, Vila Real,
Portugal) after authentication. Immediately after the harvest, the plant material was rinsed with
distilled water, weighted, and frozen (
−
20
◦
C). Plants were lyophilized (Dura Dry TM
µ
P freeze-drier;
−45 ◦C, 250 mTorr) and then were conveniently stored.
Antioxidants 2020,9, 482 4 of 20
2.3. Preparation of Extracts
Freeze-dried T. zygis aerial parts were ground to a fine powder (using a coffee mill) and
then extracted according to two extraction methods: aqueous decoction (AD), aiming to mimic
human consumption as a herbal tea or condiment, and exhaustive hydroethanolic extraction (HE), a
method optimized to obtain all the extractable compounds within the plant material, as described in
Martins-Gomes et al. [
27
]. For both extraction methods, 0.5 g of lyophilized and ground plants were
used. For the AD extraction, 150 mL of distilled water were added to the plant material and boiled for
20 min, under constant stirring. The extract was filtered twice (Whatman n
◦
4 filter, Whatman, USA,
and fiberglass filter 1.2
µ
m; VWR International Ltd., Radnor, PA, USA). For the HE extraction, 50 mL of
ethanol solution (80% v/v, in water) were added to the plant fine powder. The mixture was agitated at
room temperature for one hour (orbital shaker, 150 rpm) and centrifuged (7000 rpm, 4
◦
C; for 5 min,
Sigma Centrifuges 3–30 K, St. Louis, MO, USA). After centrifugation, the supernatant was filtered
twice (Whatman n
◦
4 filter, Whatman, USA; and fiberglass filter, 1.2
µ
m, VWR International Ltd.,
Radnor, PA, USA) and collected. Then extraction of the pellet was repeated two times more as described
above. All the supernatants were combined. Both extracts were concentrated in a rotary evaporator
(35 ◦C), freeze-dried, weighted to calculate the yields, and properly stored until further analysis.
2.4. Total Phenolic Compound Content
For the determination of the total phenolic compound (TPC) contents of the extracts the
Folin–Ciocalteau method was used. To 1 mL of T. zygis extracts (0.1 mg/mL), 0.5 mL of Folin–Ciocalteau
reagent, and 1 mL of sodium carbonate (Na
2
CO
3
; 7.5 %, w/v) were added, and the volume was adjusted
to 10 mL with distilled water. The mixture was incubated (1 h, room temperature), and the absorbance
at 725 nm was read (PerkinElmer, Lambda 25 UV/VIS Spectrometer) [
28
]. Caffeic acid was used as
standard and TPC was expressed as caffeic acid equivalents (mg CA eq./g lyophilized plant or mg CA
eq./g extract) [29,30].
2.5. Total Flavonoid Content
For the determination of the total flavonoid content (TFC) of the extracts, the method described by
Jia et al. [
31
] was used. To 1 mL of T. zygis extracts solution (0.5 mg/mL), 150
µ
L of an aqueous sodium
nitrite solution (NaNO
2
; 5%, w/v) was added, and the mixture was incubated at room temperature
for 5 min. After this time, 150
µ
L of AlCl
3
solution (10 %, w/v) were added and incubated for 6 min.
Finally, 1 mL of sodium hydroxide solution (NaOH; 1 M) was added and the absorbance at 510 nm
was read. The used standard was catechin, and TFC was expressed as mg catechin equivalents (mg C
eq./g lyophilized plant or mg C eq./g extract).
2.6. Total Ortho-Diphenol Content
For the determination of the ortho-diphenol (ODP) content, the method described by Machado,
Felizardo, Fernandes-Silva, Nunes and Barros [
28
] was used. To 4 mL of the T. zygis extracts (0.1 mg/mL),
1 mL of sodium molybdate solution (Na
2
MoO
4
; 5%, w/v) was added, and the mixture was incubated at
room temperature for 15 min. The absorbance was read at 370 nm. Caffeic acid was the standard used,
and the ODP content was expressed as mg caffeic acid equivalents (mg CA eq./g lyophilized plant or
mg CA eq./g of extract).
2.7. In Vitro Antioxidant Activity Assessment
2.7.1. ABTS•+Scavenging Assay
The ABTS
•+
scavenging activity of T. zygis extracts was measured using the method described
by Machado, Felizardo, Fernandes-Silva, Nunes and Barros [
28
]. ABTS
•+
was produced by mixing
equal volumes of a 7 mM aqueous ABTS solution and a 2.45 mM solution of potassium persulfate.
Antioxidants 2020,9, 482 5 of 20
The mixture was allowed to react at room temperature in the dark for 15–16 h. After this time, the
mixture was diluted in 20 mM acetate buffer at pH 4.5, in order to obtain an absorbance at 734 nm
of 0.700
±
0.02. The scavenging activity of T. zygis extracts were measured by adding 2 mL of the
diluted ABTS
•+
solution to 200
µ
L of extracts (0.1 mg/mL). The mixture was incubated for 15 min at
room temperature, and the absorbance at 734 nm was read. The antioxidant standard used was Trolox
((
±
)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid). The ABTS
•+
scavenging activity was
expressed as Trolox equivalents (mmol Trolox/g lyophilized plant or mmol Trolox/g extract).
2.7.2. Hydroxyl Radicals Scavenging Assay
For the determination of the site-specific and non-site-specific hydroxyl radical (
•
OH) scavenging
activity, the methods described by Taghouti et al. [
32
] were used. For the site-specific assay, to 0.5 mL
of T. zygis extracts extract solution (0.1 mg/mL), 100
µ
L of deoxyribose (20 mM), 100
µ
L of iron (II)
chloride solution (FeCl
2
; 1 mM), 100
µ
L of ascorbic acid solution (1 mM), and 100
µ
L of hydrogen
peroxide (H
2
O
2
; 10 mM) were added, followed by the addition of 400
µ
L of phosphate buffer solution
(20 mM; pH 7.4). For the determination of the non-site-specific assay, the same protocol described
above was used, but with the addition of 100
µ
L of ethylenediaminetetra-acetic acid (EDTA; 1 mM).
After incubation for 1 h at 37
◦
C, 1.5 mL of a 5% TBA solution (thiobarbituric acid, prepared in
trichloroacetic acid, 10%) were added. The mixture was boiled (100
◦
C) for 15 min and the absorbance
was read at 532 nm. A reference blank was used as control, using the same protocols but replacing the
T. zygis solution with 0.5 mL of distilled water. The site-specific and non-site-specific
•
OH scavenging
activity was expressed as the percentage inhibition using Equation (1):
Inhibition (%)=Blank abs −Sample abs
Blank abs ×100 (1)
2.7.3. Nitric Oxide Radical Scavenging Assay
For the determination of the nitric oxide radical (NO
•
) scavenging activity, the method described
by Sreejayan and Rao [
33
] was performed. For the production of the NO
•
, a 5 mM sodium nitroprusside
solution in phosphate buffer (0.1 M H
3
PO
4
; pH 7.4) was oxygenated by purging with air for 15 min.
To 0.5 mL T. zygis extracts (1 mg/mL), 4.5 mL of sodium nitroprusside solution were added and the
mixture was incubated for 2 h at 35
◦
C. NO
•
was quantified using the Griess colorimetric assay. To 1 mL
of the previous mixture, 1 mL of Griess reagent (equal volumes of 1% sulfanilamide in 5% phosphoric
acid and 0.1% N-alpha-naphthyl-ethylenediamine in water) was added, and the mixture was incubated
for 3 min at room temperature. The absorbance was measured at 546 nm. Sodium nitrite was used as
the positive control and the NO
•
scavenging activity was expressed as the inhibition percentage and
calculated according to Equation (1). For the blank determination, the T. zygis extract solution was
replaced by the same volume of distilled water.
2.8. Determination of the Phenolic Profile by High Performance Liquid Chromatography with Diode Array
Detector and High Performance Liquid Chromatography with Electrospray Ionization and Tandem Mass
Spectrometry Detection
Reversed phase HPLC-DAD analysis was carried out using an Ultimate 3000 HPLC equipped
with an Ultimate 3000 pump, a WPS-3000 TSL Analyt auto-sampler and an Ultimate 3000 column
compartment coupled to a PDA-100 photodiode array detector (Dionex, Sunnyvale, CA, USA) and
HPLC-ESI-MS
n
analysis was carried out using a Thermo Scientific system equipped with a Finnigan
Surveyor Plus auto-sampler, photodiode array detector and pump, and an LXQ Linear ion trap detector
was used for LC-MS
n
analysis as previously described by Taghouti, Martins-Gomes, Schafer, Felix,
Santos, Bunzel, Nunes and Silva [32].
Individual phenolic compounds were identified based on ultraviolet-visible (UV-Vis) spectra,
retention time, and mass spectra and compared to commercial standards and/or literature data.
The calibration curves of available commercial standards were prepared for the quantification
Antioxidants 2020,9, 482 6 of 20
of individual phenolic compounds [
32
]. When no commercial standards were available,
phenolic compounds were quantified using the aglycones or standard compounds with structural
similarity. Apigenin-(?)-O-hexuronide was quantified as apigenin; luteolin-(?)-O-hexoside and
luteolin-(?)-O-hexorunide were quantified as luteolin; salvianolic acid K was quantified as
rosmarinic acid.
2.9. Quantification of Oleanolic Acid (OA) and Ursolic Acid UA) in Hydroethanolic Extracts
For the quantification of ursolic acid (UA) and oleanolic acid (OA) in the HE extracts, the
RP-HPLC (ACE 5 C18 column; 250 mm
×
4.6 mm; particle size 5
µ
m) method, described in [
27
], was
used. The separation was performed using sodium phosphate buffer (30 mM, pH 3) as solvent A,
and methanol as solvent B, and during the run, the temperature was held at 40
◦
C. The identification
of OA and UA was performed by UV-VIS spectra (200 to 400 nm) and the retention time of the
commercial standards. Quantification was performed using the calibration curves of the UA and OA
commercial standards.
2.10. In Vitro Cell-Based Assays
2.10.1. Cell Maintenance and Handling
In this study, two human cell lines: Caco-2 (human colon adenocarcinoma cell line; Cell Lines
Service, Eppelheim, Germany) and HepG2 (human hepatocellular carcinoma cell line; ATCC
®
Number:
HB-8065TM, a gift from Prof. C. Palmeira CNC-UC, Portugal) and a mouse cell line: RAW 264.7
(mouse macrophages, Abelson murine leukemia virus-induced tumor cell line; Cell Lines Service,
Eppelheim, Germany) cells were used to evaluate the anti-proliferative and anti-inflammatory activities
of T. zygis extracts. Cells were cultured in complete culture media (Dulbecco’s Modified Eagle Media
(DMEM), supplemented with 1 mM L-glutamine, 10% (v/v) fetal bovine serum (FBS), and antibiotics
(penicillin at 100 U/mL, and streptomycin at 100
µ
g/mL) and maintained in incubator (5% CO
2
/95% air;
37
◦
C, controlled humidity). Near-confluence, Caco-2 and HepG2 cells were sub-cultured by using
an enzymatic (trypsin-EDTA) treatment (for 8 and 6 min, respectively for Caco-2 and HepG2 cells),
which was stopped using the complete culture medium (1:1, trypsin:culture media), or in the case of
RAW 264.7 cells, which were scratched offfrom the flasks using a cell scratcher (Orange Scientific;
Braine-L’Alleud, Belgium). In both cases, cells were gently re-suspended using a Pasteur pipette,
counted using an automated cell counter (TC10
™
, BIORAD, Portugal), and then re-suspended in
fresh culture media to achieve a final density of 5
×
10
4
cells/mL. Cells were then seeded into 96-well
microplates (100
µ
L/well; of 5
×
10
4
cells/mL), maintained in an incubator, and allowed to adhere and
stabilize for 48 h, for other details see [34–36].
2.10.2. Cell Viability/Cytotoxicity or Anti-Proliferative Activity Assay
The Alamar Blue assay
®
[
35
] was used to assess the anti-proliferative effect of the extracts. Stock
solutions (10 mg/mL) of T. zygis extracts were prepared in PBS for the AD extract, and 10% DMSO
(in PBS) for the HE extract. The DMSO final concentration, in test solutions, was never higher than
1%. After the cell adherence and stabilization period, culture media was removed and replaced
with test solutions (100
µ
L/well) and prepared by dilution of respective stock solutions in FBS-free
culture medium (range 50–750
µ
g/mL for Caco-2 and HepG2, and 10–200
µ
g/mL for RAW 264.7).
After 24 h or 48 h of the cell’s exposure to extracts, test solutions were removed (by gently pipette
aspiration), and immediately replaced by Alamar Blue solution (100
µ
L/well; at 10% (v/v), in FBS-free
culture medium). Absorbance was read, after 5 h incubation, at 570 nm (resorufin; reduced form) and
620 nm (resazurin; oxidized form) using a microplate reader (Multiskan EX; MTX Lab Systems, Inc.,
Bradenton, FL, USA). In each assay, a control was performed, consisting of non-treated cells (positive
control) and Alamar Blue solution alone (negative control). In the positive control, cells were submitted
to all procedures (i.e., replacing of media (with only FBS-free culture media), Alamar Blue solution
Antioxidants 2020,9, 482 7 of 20
exposure) simultaneously with the cell’s exposure to the test solutions. The results are expressed as
cell viability (% of control; i.e., positive control), calculated as described by Andreani, et al. [35].
2.10.3. Anti-Inflammatory Activity
In this work, RAW 264.7 cells were used to assess the anti-inflammatory activity of T. zygis extracts,
as described by Carbone et al. [
34
]. Briefly, RAW 264.7 cells previously seeded in 96-well plates
(100
µ
L/well, at 5
×
10
4
cells/mL), with a stabilization period of 48 h after seeding, were incubated with
various concentrations of non-cytotoxic T. zygis extract concentrations (see results) in the presence and
in the absence of lipopolysaccharide (LPS; at 1
µ
g/mL). LPS induces the production of nitric oxide
(NO) that is released into the incubation media. After incubation (24 h) with extracts from each well,
50
µ
L of each well supernatant was transferred into a new 96-well plate and, subsequently, 50
µ
L of
Griess reagent [1% (w/v) sulfanilamide prepared in 5% (w/v) H
3
PO
4
(v/v) and 0.1% (w/v)N-(1-naphthyl)
ethylenediamine dihydrochloride in water] were added to each well. After 15 min of incubation time
(room temperature, under dark), the absorbance at 550 nm was read (Multiskan EX microplate reader;
MTX Lab Systems, Inc., Bradenton, FL, USA). Quantification was performed against a standard curve
calculated with sodium nitrite (NaNO
2
; in the range 0 to 100
µ
M) and the results were expressed as %
of control (i.e., nitrite production by the positive control cells (LPS-stimulated cells in the absence of
T. zygis extracts) set to 100%, that is, 0% of anti-inflammatory effect.
2.11. Data and Statistical Analysis
For each extraction method, three individual extractions were performed, and the analyses were
performed in triplicate for all the assays. The IC
50
values for the anti-proliferative activity were
calculated as described by Silva et al. [
37
]. Significant differences for the phenolic composition and
antioxidant activity were performed using the t-Student test (
α
=0.05). For the comparison of the IC
50
values for the anti-proliferative activity and anti-inflammatory activity, analyses of variance (ANOVA)
followed by Tukey’s multiple test (
α
=0.05) were performed (GraphPad Prism version 7, GraphPad
Software Inc., San Diego, CA, USA).
3. Results and Discussion
3.1. Extract Yield and Chemical Composition of T. zygis Extracts
In this study, two extraction methods were used to obtain T. zygis subsp. zygis (T. zygis) extracts:
an exhaustive hydroethanolic (HE) extraction and the aqueous decoction (AD). The HE was previously
shown to extract 99% of the total extractable compounds [
27
], thus it was chosen as the method to
obtain the full “free” phenolic composition of T. zygis subsp. zygis. The AD extraction mimics the
common procedure of beverage preparation. Therefore, it allows to analyze the phenolic compounds
that are available with a common preparation for human consumption, as these plants are also used as
herbal teas, seasoning, and condiments.
Table 1shows that the yield of T. zygis subsp. zygis AD extract is higher than the one of the HE
extract (~28% higher, p<0.05), denoting differences in the extraction yield between the HE and AD
extraction methods. Concerning the AD extract, the yield obtained in this work (29.70
±
0.99 %, Table 1)
is higher than that described for T. zygis (subspecies not mentioned) by Afonso et al. (2018), who
reported an extraction yield of 12%. Nevertheless, the extraction conditions were significantly different
(5 g plant/100 mL of water, for 15 min) from that used in this work. The higher yield values of T. zygis
extracts (Table 1), compared to other species produced in the same place (such as Thymus carnosus [
27
],
Thymus pulegioides [
32
], T. mastichina [
38
] and T. vulgaris [
39
]), might result from a species effect or from
the time of year in which they were harvested, as T. zygis was harvested in April (blooming stage) and
the other ones in October (post-blooming, end fructification stage), the latter hypothesis still needs to
be confirmed with more experimental data and other Thymus species harvested in the same place at
both stages.
Antioxidants 2020,9, 482 8 of 20
Table 1.
Thymus zygis subsp. zygis extracts: extraction yields, chemical composition, and
antioxidant activity.
Hydroethanolic Extract Aqueous Decoction
Extraction yield (%, w/w) 22.83 ±0.96 29.70 ±0.99 *
Total phenols (mg Caffeic acid eq./g) Ext. 195.81 ±7.07 97.31 ±7.67 *
D.P. 44.70 ±1.61 28.90 ±2.28 *
Total flavonoids (mg Catechin eq./g) Ext. 269.49 ±10.39 124.80 ±11.62 *
D.P. 61.52 ±2.37 37.07 ±3.45 *
Ortho-diphenols (mg Caffeic acid eq./g) Ext. 139.79 ±1.28 78.55 ±0.80 *
D.P. 31.91 ±0.29 23.33 ±0.24 *
ABTS•+(mmol Trolox eq./g) Ext. 1.08 ±0.15 0.76 ±0.14
D.P. 0.25 ±0.03 0.23 ±0.04
•OH radicals +EDTA (% inhibition) 66.28 ±1.20
•OH radicals −EDTA (% inhibition) 43.15 ±2.88
NO•radicals
(% inhibition, after 120 min)
29.32 ±1.67
Abbreviations: D.P., dry plant; Ext., extract. In antioxidant activity, the percentage of inhibition was obtained for
1 mg/mL of extract. Significant statistical differences between extraction methods (*) when (p<0.05).
T. zygis subsp. zygis TPC contents obtained with the HE extraction method were significantly
higher than that extracted with the AD extraction procedure (Table 1). The HE extract yielded about
twice the TPC contents of the AD extract (HE: 195.81
±
7.07 and AD: 97.31
±
7.67 mg CA eq./g extract);
but we also observe that, although not exhaustive, AD extraction extracts about 65% of plant’s TPC
(Table 1; HE: 44.70 ±1.61 and AD: 28.90 ±2.28 mg CA eq./g D.P.).
Comparing the TPC content per gram of dry plant, in plants collected in the same place and
extracted with the same HE extraction method, we observed an order for TPC contents (in mg CA
eq./g D.P.), T. carnosus (84.4 [
27
]) >> T. zygis subsp. zygis (44.7, Table 1) ~ T. pulegioides (43.0 [
32
]) >>
Thymus citriodorus (27.7 [
39
])
≥
T. vulgaris (25.12 [
39
]) ~ T. mastichina (24.6 [
38
]). Concerning the AD
extraction, the same trend was observed, being the TPC contents (in mg CA eq./g D.P.) of T. carnosus
(54.5 [
27
]) >> T. zygis subsp. zygis (28.9, Table 1)>T. pulegioides (26.1 [
32
]) >T. vulgaris (21.6 [
39
]) >> T.
citriodorus (15.5 [
39
]) >T. mastichina (12.5 [
38
]). Methanolic extracts of T. zygis, harvested in several
locations in Spain, were also reported to have high TPC contents [
17
] in identical amounts as the here
reported (Table 1). These data indicate that T. zygis is a suitable source of phenolic compounds.
For the TFC extracted from T. zygis subsp. zygis, the results obtained are in line with those
described for the TPC. The amount of TFC extracted by HE extraction was significantly higher than
those obtained by AD extraction (Table 1). For the ODP, as observed for the TFC and TPC, the levels
present in the HE extract were significantly higher than that obtained in the AD extract (Table 1). These
data highlight the value of the T. zygis subspecies zygis as a thyme species with high content in potential
bioactive molecules.
3.2. T.zygis subps. zygis Aqueous Decoction and Hydroethanolic Extracts Phenolic Profiles
In order to have a deeper understanding of the chemical composition of T. zygis subsp. zygis
and the relation with its extracts bioactivities, the phenolic composition of HE and AD extracts was
determined by HPLC-DAD and HPLC-MS
n
. The phenolic profile of the HE and AD extracts, as well
as their concentrations, is shown in Figure 1and in Table 2.
Antioxidants 2020,9, 482 9 of 20
Antioxidants 2020, 9, x FOR PEER REVIEW 9 of 21
Figure 1. Phenolic profile of hydroethanolic (HE) (A) and aqueous decoction (AD) (B) extracts of
Thymus zygis subsp. zygis, obtained by High-performance liquid chromatography, coupled to diode
array detector (HPLC-DAD). For peak number identification, please refer to Table 2.
Figure 1.
Phenolic profile of hydroethanolic (HE) (
A
) and aqueous decoction (AD) (
B
) extracts of
Thymus zygis subsp. zygis, obtained by High-performance liquid chromatography, coupled to diode
array detector (HPLC-DAD). For peak number identification, please refer to Table 2.
Antioxidants 2020,9, 482 10 of 20
Table 2.
Phytochemical composition of Thymus zygis subsp. zygis hydroethanolic (HE) and aqueous decoction (AD) extracts determined by high performance liquid
chromatography coupled to diode array detector and electrospray ionization mass spectrometry.
Compound R.T. (min) ESI-MS2
Quantification
HE AD E.
M.
E.
mg/g D.P. mg/g Extract mg/g D.P. mg/g Extract
1Salvianic acid A 18.48 ±0.22 [197] n.q. n.q. 0.17 ±0.01 0.58 ±0.4
2Eriodictyol-di-O-hexoside 21.81 ±0.19 [611]:449;287 n.q. n.q. n.d. n.d.
3Chlorogenic acid 23.72 ±0.21 [353]:191;179;173;135 n.q. n.q. n.d. n.d.
4Unknown 24.72 ±0.22 [563]:545;517;455 n.q. n.q. n.q. n.q.
5Hydroxyjasmonic acid–hexoside 24.89 ±0.10 [387]:369;225;207;163 0.03 ±0.01 0.14 ±0.03 0.31 ±0.01 1.07 ±0.02 *
6Apigenin-(6,8)-C-diglucoside 25.02 ±0.18 [593]:575;503;473;383 353 0.17 ±0.04 0.76 ±0.15 0.56 ±0.03 1.85 ±0.1 *
7Caffeic acid 25.52 ±0.22 [179]:135 0.08 ±0.02 0.36 ±0.09 0.56 ±0.01 1.89 ±0.02 *
8Unknown 26.05 ±0.19 [495]:486;451;375;368 n.q. n.q. n.q. n.q.
9Eriodictyol-(?)-O-hexoside 26.16 ±0.23 [449]:287 2.00 ±0.26 8.77 ±1.13 1.43 ±0.05 4.80 ±0.17 *
10 Unknown 27.95 ±0.95 [367]:193;173;155;137;111 n.q. n.q. n.q. n.q.
11 Prolithospermic acid 28.73 ±0.25 [357]:313;269;245;203 n.d. n.d. n.q. n.q.
12 Naringenin-O-hexoside 29.4 ±0.26 [433]:313;271;267;137 n.d. n.d. n.q. n.q.
13 Quercetin-(?)-O-hexoside 30.48 ±0.04 [463]:301 0.92 ±0.17 4.05 ±0.76 0.39 ±0.06 1.31 ±0.22
14 Naringenin-O-hexoside 30.88 ±0.34 [433]:313;271 n.q. n.q. n.q. n.q.
15 Luteolin-O-hexoside 32.74 ±0.41 [447]:285 0.83 ±0.12 3.64 ±0.51 0.70 ±0.05 2.38 ±0.16 *
16 Luteolin-(?)-O-rutinoside 33.87 ±0.30 [593]:285 n.q. n.q. n.q. n.q.
17 Quercetin-(?)-O-hexuronide 34.42 ±0.28 [477]:301 n.q. n.q. n.q. n.q.
18 Luteolin-(?)-O-hexoside 34.84 ±0.30 [447]:285 4.44 ±0.57 19.46 ±2.49 4.23 ±0.30 14.23 ±1.00
19 Salvianolic acid B/E isomer 2 37.30 ±0.28 [717]:555;519;475;357;295 n.q. n.q. n.q. n.q.
20 Quercetin-(?)-acetyl-hexoside 38.06 ±0.38 [549]:531;505;486;416;345;301 1.03 ±0.22 4.52 ±0.87 0.98 ±0.15 3.30 ±0.5
21 Salvianolic acid A isomer 38.46 ±0.34 [493]:383;313;295 n.q. n.q. n.d. n.d.
22 Luteolin-(?)-O-hexorunide 38.77 ±0.34 [461]:285;175 2.92 ±0.32 12.78 ±1.40 3.01 ±0.32 10.14 ±1.06
23 Chrysoeriol-(?)-O-hexoside 39.90 ±0.33 [461]:299;160 n.q. n.q. n.q. n.q.
24 Rosmarinic acid 39.44 ±0.38 [359]:223;179;161 11.11 ±1.39 48.65 ±5.34 4.18 ±0.78 14.07 ±2.62 *
25 Salvianolic acid I 41.37 ±0.88 [537]:493;448;359;339;313;295 3.31 ±0.48 14.52 ±2.10 0.95 ±0.20 3.21 ±0.65 *
26 Salvianolic acid K 42.33 ±0.30 [555]:537;493;359 2.36 ±0.37 10.33 ±1.62 2.11 ±0.30 7.10 ±1.03
27
Quercetin-(?)-O-hexoside-hexuronide
43.36 ±0.38 [639]:301 n.q. n.q. n.q. n.q.
28 Apigenin-(?)-O-hexuronide 44.73 ±0.44 [445]:269;175 n.q. n.q. n.q. n.q.
29 Chrysoeriol-(?)-O-hexuronide 45.86 ±0.30 [475]:299 n.q. n.q. n.q. n.q.
30 Unknown 49.50 ±0.44 [551]:519;359;339;313;221;179 n.q. n.q. n.d. n.d.
31 Oleanolic acid a35.85 ±0.05 - 0.22 ±0.03 0.99 ±0.15 n.d. n.d.
32 Ursolic acid a36.91 ±0.05 - 0.48 ±0.08 2.17 ±0.35 n.d. n.d.
Total phenolic compounds 29.22 ±3.47 127.98 ±15.20 19.58 ±2.25 65.93 ±7.56 *
Total phenolic acid 16.90 ±2.03 74.00 ±8.92 8.30 ±1.29 27.93 ±4.35 *
Total flavonoids 12.32 ±1.44 53.98 ±6.31 11.28 ±0.96 37.99 ±3.21
Abbreviations: AD: aqueous decoction; HE: hydroethanolic extractions; RT: retention time; ESI-MS
2
-Fragment ions obtained after fragmentation of the pseudo-molecular ion [M]
−
; n.q.: not
quantified (but detected); n.d.: not detected; E.M.E.: extraction method effect.
a
—identified and quantified by a different method. (*) denotes significant statistical differences (t-Student
test), between extraction methods, for mg/g dry plant (D.P.), if (p<0.05). Results, from n =3 different extractions, per extract, are presented as mean ±standard deviation.
Antioxidants 2020,9, 482 11 of 20
The relative amount of phenolic compounds determined by HPLC-DAD is consistent with the
results obtained by colorimetry, and with the TPC, TFC, and OPD contents (Table 1). For the HE
extracts of T. zygis subsp. zygis, rosmarinic acid was the most abundant phenolic compound (Table 2).
Rosmarinic acid represented 38% of the total phenolic compounds extracted by the HE solution. In
the AD extract, the most abundant polyphenol was luteolin-(?)-O-hexoside (compound 18; Table 2),
accounting for 22% of the total phenolic compounds extracted, followed by rosmarinic acid (21%), and
luteolin-(?)-O-hexuronide (15%). The amount of rosmarinic acid extracted by AD method represents
only 38% of the rosmarinic acid extracted by HE. Thymus species are usually characterized by high
content of rosmarinic acid [
40
]. Taking into account that the exhaustive HE extraction method [
27
]
reflects the plant’s total extractable phenolic composition, we observe that T. zygis subsp. zygis is
also characterized by high contents of rosmarinic acid (Table 2). The HE extracts of other Thymus
species also revealed high contents of rosmarinic acid (as % of total phenolic acids), as is the case
of T. citriodorus (51% [
39
]), T. mastichina (33% [
38
]), T. vulgaris (70% [
39
]), T. pulegioides (48% [
32
]).
In contrast, other species, such as in T. carnosus contain lower amounts of rosmarinic acid (17% [
27
]).
Rosmarinic acid was also quantified in high amounts in aqueous extracts of T. zygis (52%, subspecies
not mentioned; [
19
]), [
16
]) and in Thymus algeriensis (45% [
41
]). Rosmarinic acid was indicated as
the major phenolic compound in methanolic extracts of T. zygis (subspecies not mentioned; [
16
]).
This Thymus differs from the previously mentioned Thymus species by the presence of significant
amounts of flavonoids (42% and 57% of the total phenolic compounds in the HE and AD extracts,
respectively), especially Luteolin-(?)-O-hexoside that represents 15% of the total phenolic compounds
of the HE extract, was present in lower amounts in T. vulgaris,T. citriodorus,T. carnosus,T. pulegioides
and T. mastichina [
27
,
32
,
38
]. In fact, T. zygis subspecies zygis is the Thymus species studied by our group
that contains the second highest levels of flavonoids quantified in the HE extracts (T. pulegioides (61%),
T. mastichina (39%), T. citrodorus (24%), T vulgaris (16%), and T. carnosus (6%)). The AD extraction
allowed to recover higher amounts of caffeic acid when compared to the HE extraction. This higher
amount of caffeic acid in the AD extraction can be due to the hydrolysis of rosmarinic acid during the
AD extraction that is performed with hot water. This hypothesis is supported by the fact that in the
AD extracts higher amounts of salvianic acid A were also present (Table 2).
The most abundant phenolic compounds described for T. zygis subsp. zygis (Table 2) are in
agreement with those described by Afonso et al. (2018) for aqueous extracts of Thymus zygis (harvested
in the same location), although the relative amounts found were different, which might have resulted
from extraction procedure (different from the ones in current work), harvesting period, or the use of a
different Thymus zygis subspecies (not disclosed).
3.3. Oleanolic Acid and Ursolic Acid Contents
T. zygis subsp. zygis HE extracts contained OA and UA (Table 2), however, their levels were low
(0.26 and 0.55 mg/g dry plants) compared to other thyme species (T. serpyllum (3.7 and 13.9 mg/g dry
plant, or OA and of UA, respectively [
42
]), T. carnosus (9.9 and 18.7 mg/g dry plant, of OA and of UA
respectively [
27
]) and T. pulegioides (0.34 and 0.80 mg/g dry plant, of OA and of UA, respectively [
32
]).
These differences may reflect a different phenological state of the plant, the effect of location, and of the
climate on the chemical composition of the plants. To the best of our knowledge, this is the first report
in which UA and OA are described in T. zygis extracts. However, the presence of the diterpene carnosic
acid was described in extracts of Thymus zygis subsp. gracilis shrubs (cultivated in Spain), obtained
with petroleum ether and methanol, in amounts of ~120
µ
g/g dry plant [
18
]. The chromatogram of
T. zygis subsp. zygis HE extract, shown in Figure 2, reveals the presence of OA and UA, as compared
by the chromatograms of OA and UA standards (two upper traces, as denoted).
Antioxidants 2020,9, 482 12 of 20
Antioxidants 2020, 9, x FOR PEER REVIEW 13 of 21
Figure 2. Chromatogram of oleanolic acid (OA) and ursolic acid (UA) standards and of Thymus zygis
hydroethanolic (HE) extract.
3.4. In Vitro Antioxidant Activity
The HE extracts of T. zygis subsp. zygis (at 1 mg/mL) showed higher ABTS+• radical scavenging
activity (~0.25 Trolox eq./g dry plant, Table 1) in relation to the AD extracts (~0.23 mmol Trolox eq./g
dry plant, Table 1). The values obtained for the HE extracts (Table 1) were lower than those found for
T. pulegioides HE extracts (0.34 mmol Trolox eq./g D.P.; [32] but in the same range of T. vulgaris and T.
citriodorus (0.22 mmol Trolox eq./g D.P.; [39]), T. carnosus (0.21 mmol Trolox eq./g D.P.; [27]) and T.
mastichina (0.20 mmol Trolox eq./g D.P.; [38]). In contrast, the ABTS•+ radical scavenging activity of
AD extracts of T. zygis subsp. zygis (0.21 mmol Trolox eq./g D.P.; Table 1) was higher than that
described for the other Thymus species (T. pulegioides 0.15 mmol Trolox eq./g D.P. [32]; T. carnosus
0.14 mmol Trolox eq./g D.P. [27]; T. mastichina 0.08 mmol Trolox eq./g D.P.[38] and T. citriodorus (0.11
mmol Trolox eq./g dry plant [39]), being similar to that found for AD extracts of T. vulgaris (0.20
mmol Trolox eq./g dry plant; [39]). Relevant antioxidant activity was also reported for water extracts
of T. zygis (ABTS radical scavenging with IC50 = 15.43 µg/mL; subspecies not mentioned) collected in
the southeastern Morocco [21] and for petroleum ether/methanolic extract of T. zygis subsp. gracilis
(DPPH radical scavenging with IC50 = 3.7 µg/mL) harvested in Murcia, Spain [18].
T. zygis subsp. zygis AD extract (at 1 mg/mL) exhibited a higher non-site-specific inhibition
activity when compared to the site-specific inhibition activity (Table 1). In the non-site-specific assay
we evaluated the efficiency of the compounds present in the extracts to compete with deoxyribose
for •OH radicals that are produced by the Fe2+-EDTA chelate. On the other hand, for the site-specific
assay, when EDTA is not present in the reaction mixture, the Fe3+ can bind directly to deoxyribose
and produce •OH. Ribose degradation inhibition, in the absence of EDTA, indicates the iron ion
chelating possibility and also the trapping of the •OH radical. Therefore, the competition of the
compounds present in the extract for scavenging the •OH seems to be the main mechanism,
although compounds present in the extract can also effectively bind the Fe3+ ions [43,44]. Compared
with other works, an IC50 value of 3.7 mg/mL was reported for an aqueous extracts of T. vulgaris [45],
a value much higher than that described here. On the other hand, Chung et al. [46] reported more
than 75% inhibition of ribose degradation for a thyme methanolic extract (1 µg/mL; unspecified
species). The HO• radical scavenging values obtained for T. zygis subsp. zygis AD extracts (Table 1)
show that this thyme species has a higher inhibition capacity against hydroxyl radical compared to
T. carnosus (41% [27]), T. pulegioides (34% [32]), T. mastichina (49% [38]), T. citriodorus (38% [39]) and T.
vulgaris (10% [39]), when the assay was performed in the presence of EDTA. In the absence of EDTA,
Figure 2.
Chromatogram of oleanolic acid (OA) and ursolic acid (UA) standards and of Thymus zygis
hydroethanolic (HE) extract.
3.4. In Vitro Antioxidant Activity
The HE extracts of T. zygis subsp. zygis (at 1 mg/mL) showed higher ABTS
+•
radical scavenging
activity (~0.25 Trolox eq./g dry plant, Table 1) in relation to the AD extracts (~0.23 mmol Trolox eq./g
dry plant, Table 1). The values obtained for the HE extracts (Table 1) were lower than those found
for T. pulegioides HE extracts (0.34 mmol Trolox eq./g D.P.; [
32
] but in the same range of T. vulgaris
and T. citriodorus (0.22 mmol Trolox eq./g D.P.; [
39
]), T. carnosus (0.21 mmol Trolox eq./g D.P.; [
27
]) and
T. mastichina (0.20 mmol Trolox eq./g D.P.; [
38
]). In contrast, the ABTS
•+
radical scavenging activity of
AD extracts of T. zygis subsp. zygis (0.21 mmol Trolox eq./g D.P.; Table 1) was higher than that described
for the other Thymus species (T. pulegioides 0.15 mmol Trolox eq./g D.P. [
32
]; T. carnosus 0.14 mmol
Trolox eq./g D.P. [
27
]; T. mastichina 0.08 mmol Trolox eq./g D.P. [
38
] and T. citriodorus (0.11 mmol Trolox
eq./g dry plant [
39
]), being similar to that found for AD extracts of T. vulgaris (0.20 mmol Trolox eq./g
dry plant; [
39
]). Relevant antioxidant activity was also reported for water extracts of T. zygis (ABTS
radical scavenging with IC50 =15.43 µg/mL; subspecies not mentioned) collected in the southeastern
Morocco [
21
] and for petroleum ether/methanolic extract of T. zygis subsp. gracilis (DPPH radical
scavenging with IC50 =3.7 µg/mL) harvested in Murcia, Spain [18].
T. zygis subsp. zygis AD extract (at 1 mg/mL) exhibited a higher non-site-specific inhibition activity
when compared to the site-specific inhibition activity (Table 1). In the non-site-specific assay we
evaluated the efficiency of the compounds present in the extracts to compete with deoxyribose for
•
OH radicals that are produced by the Fe
2+
-EDTA chelate. On the other hand, for the site-specific
assay, when EDTA is not present in the reaction mixture, the Fe
3+
can bind directly to deoxyribose and
produce
•
OH. Ribose degradation inhibition, in the absence of EDTA, indicates the iron ion chelating
possibility and also the trapping of the
•
OH radical. Therefore, the competition of the compounds
present in the extract for scavenging the
•
OH seems to be the main mechanism, although compounds
present in the extract can also effectively bind the Fe
3+
ions [
43
,
44
]. Compared with other works,
an IC
50
value of 3.7 mg/mL was reported for an aqueous extracts of T. vulgaris [
45
], a value much higher
than that described here. On the other hand, Chung et al. [
46
] reported more than 75% inhibition of
ribose degradation for a thyme methanolic extract (1
µ
g/mL; unspecified species). The HO
•
radical
scavenging values obtained for T. zygis subsp. zygis AD extracts (Table 1) show that this thyme
species has a higher inhibition capacity against hydroxyl radical compared to T. carnosus (41% [
27
]),
Antioxidants 2020,9, 482 13 of 20
T. pulegioides (34% [
32
]), T. mastichina (49% [
38
]), T. citriodorus (38% [
39
]) and T. vulgaris (10% [
39
]), when
the assay was performed in the presence of EDTA. In the absence of EDTA, the T. zygis subsp. zygis AD
extract presented significant inhibition capacity of the
•
OH (43%, Table 1), too, similar to that observed
for AD extracts of T. carnosus (41% [
27
]) but higher than that observed for T. pulegioides (31% [
32
]), T.
mastichina (28% [
38
]), T. citriodorus (31% [
39
]) and T. vulgaris (21% [
39
]) AD extracts. T. zygis subsp. zygis
AD extracts produced an inhibition percentage of the
•
OH lower than that reported of the methanolic
extract of Thymus dacicus (50% radical scavenging, at 18.85 µg/mL, [47]).
Concerning the scavenging of the NO radical, T. zygis subsp. zygis AD extract (29%, Table 1)
showed lower scavenging activity than AD extracts of T. citriodorus (41%; [
39
]), T. vulgaris (58%; [
39
]),
T. carnosus (42%; [27]), T. pulegioides (35.76 %; [32]), and T. mastichina (39%; [38]).
3.5. Anti-Proliferative Effect of T. zygis subsp. zygis Extracts
The anti-proliferative activity of T. zygis subsp. zygis AD and HE extracts was assessed using the
Alamar Blue (AB) assay and the two selected cell lines, HepG2 and Caco-2. Cells were incubated with
different concentrations of T. zygis subsp. zygis extracts (50, 100, 200, 500, and 750
µ
g/mL) during 24
or 48 h. Results of anti-proliferative assay were compared with positive control cells (non-exposed
cells) and are shown in Figure 3. For the AD extract, a reduced anti-proliferative effect was observed in
both cell lines (Figure 3A: Caco-2 and 3B: HepG2). As shown, the T. zygis subsp. zygis AD extract does
not reduce HepG2 cells viability for concentrations up to 500
µ
g/mL (cell viability ~100% of control
at 24 and 48 h), however, at 500
µ
g/mL, it produces a slight reduction of Caco-2 cells viability (cell
viability was 92
±
2% and 81
±
1%, at 24 and 48 h, respectively). Although, the effect of T. zygis subsp.
zygis AD extract effect is identical on both Caco-2 and HepG2 cells, with close IC
50
values (Table 3),
the differences are statistically different, at both exposure times (p<0.05).
T. zygis subsp. zygis HE extract showed a higher cytotoxic/anti-proliferative effect than the AD
extract, in both cell lines (Figure 3B: Caco-2 and 3D: HepG2). T. zygis subsp. zygis HE extract is non-toxic
at 50
µ
g/mL (both cell lines, both exposure times). Using the HE extract at 100
µ
g/mL, only the 24 h
exposure may be considered non-toxic (cell viability was 92.3
±
3.4% and 85.8
±
7.9%, for Caco-2 and
HepG2, respectively; p<0.05), while the 48 h exposure is toxic (cell viability was 13.6
±
1.7% and
29.4
±
9.3%, for Caco-2 and HepG2, respectively; p<0.05). The HE extract at concentrations higher
than 200
µ
g/mL reduces cell viability to values below 20% of control (both cell lines, both exposure
times). As the human consumption of this plant (herbal tea, seasoning or condiments) is mimicked by
the AD extract effect, the results indicate that T. zygis subsp. zygis is non-toxic. However, the high
anti-proliferative activity/cytotoxicity of HE extracts on Caco-2 cells (IC
50
85.01
±
15.10
µ
g/mL, at 48 h
exposure, Table 3) and on HepG2 cells (IC
50
82.19
±
2.46
µ
g/mL, at 48 h exposure, Table 3) makes this
species a good source for bioactive molecules with anti-proliferative activity. This effect correlates with
its higher concentration in phenolic compounds in HE extract (Tables 1and 2), and most probably it is
due to the presence of UA and OA. The anti-proliferative activity of these triterpenoids against several
tumor cell lines is documented in several works [
48
,
49
] OA exerted strong cytotoxic effect against HT29
cells (colon adenocarcinoma) with EC
50
=5.6
µ
M [
50
], and UA and OA exerted significant anti-tumor
activity against HCT15 cells (human colon carcinoma cell line) by inhibiting cell proliferation through
cell-cycle arrest [
49
]. Among the Thymus species studied in our laboratory, T. carnosus contains higher
contents of UA and OA in its HE extract [
27
], and produced lower IC
50
values in both cell lines
(Caco-2 ~32
µ
g/mL, and HepG2 ~120
µ
g/mL, at 24 exposure). However, we may not exclude the
effect of the other compounds that may synergistically affect this anti-proliferative activity (Figure 3,
Table 3). Additionally, rosmarinic acid, the major phenolic acid in this extracts (Table 1) has been
widely described to produce anti-proliferative activity in several cell models, by mechanisms that
involve apoptosis regulation and cell-cycle arrest in sub-G1 and G2/M phases [
51
,
52
]. Salvianolic
acids have been widely described to control cellular pathways involved in cell proliferation and in
cellular migration, which are intrinsically related to cancer progression [
53
,
54
], although most of the
reported activities are to salvianolic acids A and B, salvianolic acids K and I (Table 2) might share
Antioxidants 2020,9, 482 14 of 20
structurally-related activities. The overall response of cells to T. zygis subsp. zygis extracts results from
the combined activity of all components.
Antioxidants 2020, 9, x FOR PEER REVIEW 15 of 21
Figure 3. Anti-proliferative effect of Thymus zygis subsp. zygis aqueous decoction (AD) and
hydroethanolic (HE) extracts on Caco-2 (A and B for AD and HE extracts, respectively) and HepG2
cells (C and D for AD and HE extracts, respectively). Two exposure times, 24 and 48 h, were
considered, as indicated. Results are expressed as (mean ± SD, n = 4). Statistically significant
differences (p < 0.05) between the control and sample concentrations at respective incubation time are
denoted by *, and those between exposure periods at the same concentration are denoted by #.
T. zygis subsp. zygis HE extract showed a higher cytotoxic/anti-proliferative effect than the AD
extract, in both cell lines (Figure 3B: Caco-2 and 3D: HepG2). T. zygis subsp. zygis HE extract is
non-toxic at 50 µg/mL (both cell lines, both exposure times). Using the HE extract at 100 µg/mL, only
the 24 h exposure may be considered non-toxic (cell viability was 92.3 ± 3.4% and 85.8 ± 7.9%, for
Caco-2 and HepG2, respectively; p < 0.05), while the 48 h exposure is toxic (cell viability was 13.6 ±
1.7% and 29.4 ± 9.3%, for Caco-2 and HepG2, respectively; p < 0.05). The HE extract at concentrations
higher than 200 µg/mL reduces cell viability to values below 20% of control (both cell lines, both
exposure times). As the human consumption of this plant (herbal tea, seasoning or condiments) is
mimicked by the AD extract effect, the results indicate that T. zygis subsp. zygis is non-toxic.
However, the high anti-proliferative activity/cytotoxicity of HE extracts on Caco-2 cells (IC50 85.01 ±
15.10 µg/mL, at 48 h exposure, Table 3) and on HepG2 cells (IC50 82.19 ± 2.46 µg/mL, at 48 h
exposure, Table 3) makes this species a good source for bioactive molecules with anti-proliferative
activity. This effect correlates with its higher concentration in phenolic compounds in HE extract
(Tables 1 and 2), and most probably it is due to the presence of UA and OA. The anti-proliferative
activity of these triterpenoids against several tumor cell lines is documented in several works [48,49]
OA exerted strong cytotoxic effect against HT29 cells (colon adenocarcinoma) with EC50 = 5.6 µM
Figure 3.
Anti-proliferative effect of Thymus zygis subsp. zygis aqueous decoction (AD) and
hydroethanolic (HE) extracts on Caco-2 (
A
and
B
for AD and HE extracts, respectively) and HepG2
cells (
C
and
D
for AD and HE extracts, respectively). Two exposure times, 24 and 48 h, were considered,
as indicated. Results are expressed as (mean
±
SD, n =4). Statistically significant differences (p<0.05)
between the control and sample concentrations at respective incubation time are denoted by *, and
those between exposure periods at the same concentration are denoted by #.
Table 3.
Effect Thymus zygis subsp. zygis extracts on Caco-2 and HepG2 cells, expressed as IC
50
values.
Cells were exposed to aqueous decoction (AD) and hydroethanolic (HE).
IC50 (µg/mL) Exposure Time Effect
Extraction Method Effect
AD HE AD HE
Caco-2
24 h 746.10 ±6.35 202.20 ±5.59
* *
*
48 h 604.70 ±6.70 85.01 ±15.10 *
HepG2 24 h 719.20 ±8.65 264.90 ±10.03
* *
*
48 h 638.02 ±5.24 82.19 ±2.46 *
Results are expressed as (mean ±SD, n =4); * means statistically significant at p<0.05.
Antioxidants 2020,9, 482 15 of 20
3.6. Anti-Inflammatory Effect of T. zygis subsp. zygis Extracts
The anti-inflammatory activity of T. zygis subsp. zygis was evaluated on RAW 264.7 cells, as a
consequence of extracts capacity to decrease the lipopolysaccharides (LPS)-induced nitric oxide (NO)
release when exposed to T. zygis subsp. zygis extracts, and is shown in Figure 4. First, a cell viability
assay was performed on RAW 264.7 cells (Figure 4B) to select non-cytotoxic concentrations of T. zygis
subsp. zygis extracts. The cells were exposed to AD and HE extracts, at concentrations up to 100
µ
g/mL, for 24 h. Figure 4B shows that both extracts are not cytotoxic. Due to the slight decrease in cell
viability at 100
µ
g/mL of extracts (91.2
±
1.2% and 88.1
±
3.8%, for AD and HE extract, respectively,
not statistically different from control, p>0.05)) the concentrations selected for the anti-inflammatory
assay were up to 50
µ
g/mL (Figure 4A). Both extracts showed a dose-dependent inhibition of NO
release by LPS-stimulated RAW 264.7 cells, indicating anti-inflammatory activity. The HE extract
resulted in an about two-fold higher effect compared to the AD extract, which might be the result of
the higher content in phenolic compounds (Tables 1and 2). Anti-inflammatory activity, using the
same cell model as in this work, was reported for T. zygis subsp. sylvestris essential oils [
11
], too.
Using the inhibition of protein denaturation method to estimate anti-inflammatory activity, Hmidani
et al. [
21
] reported good anti-inflammatory activity of T. zygis subsp. gracilis aqueous extracts (T. zygis
IC
50
=133.25
µ
g/mL, and indomethacin IC
50
=86.07
µ
g/mL). Using
in vivo
models, an aqueous extract
of T. zygis subsp. gracilis was reported to cause potent anti-inflammatory activity in the mice model of
croton oil-induced ear edema and significant anti-inflammatory activity in the carrageenan-induced
paw edema rat model, in comparison with indomethacin [
20
]. The croton oil-induced ear edema mice
model was also used to evaluate the anti-inflammatory effect of Thymus broussonetii (in extracts and
fractions) revealing that the main anti-inflammatory principles were UA and OA [
55
]. OA and UA,
with a skeleton of oleanane and ursane, are considered the main responsible for the anti-inflammatory
activity exhibited by a variety of medicinal plants [
56
]. This is attributed to the inhibition of enzymes
involved in, e.g., eicosanoids production (COX, cyclooxygenase; and phospholipase A2), that results in
reducing processes inflammatory [
48
,
57
–
59
]. However, the AD extract that did not contain UA and
OA also showed significant anti-inflammatory activity. In addition, although, the presence of UA and
OA in the HE extract could justify the higher anti-inflammatory activity of the HE extract, compared to
the AD extract (Figure 4), the amount of UA and OA at 50
µ
g/mL of the HE extract was about 0.1
µ
M
(~50 ng/mL), which is very low. However, Figure 4A,B show that, at each tested concentration, the HE
extract is about two-fold more potent than the AD extract, which corresponds to the ratio of the total
phenolic compounds between the HE and AD extracts (Table 2). Therefore, it can be suggested that
other compounds that are present in higher levels in the HE extract may also contribute to the observed
potent anti-inflammatory activity of T. zygis subsp. zygis HE extract [
60
]. Thus, we may confirm and
conclude that phenolic compounds of T. zygis subsp. zygis have high anti-inflammatory potential.
Concerning the phenolic composition of these extracts and related contribution to the verified
anti-inflammatory effect, rosmarinic acid has been demonstrated to produce an anti-inflammatory effect
both in
in vitro
and
in vivo
experimental models, and by modulating several mechanisms [
40
]. In RAW
264.7 cells, rosmarinic acid was shown to inhibit inducible nitric oxide synthase (iNOS) activity [
61
],
resulting in lower NO released levels. The reduction of NO release induced by rosmarinic acid by
LPS-stimulated RAW 264.7 cells was also reported by Martins-Gomes et al. [
27
]. Recently, other Thymus
species, Thymus algeriensis, was reported to have a high content in salvianolic acid K, together with
rosmarinic acid and luteolin glucuronide, which was correlated with the reported anti-inflammatory
activity [
62
]. The role of the individual phenolic compounds of extracts, such as the salvianolic acid K
and I, in modulating specific cellular pathways involved in inflammation and proliferation are worth
of further study aiming at the discovery of novel pharmacological relevant molecules.
Antioxidants 2020,9, 482 16 of 20
Antioxidants 2020, 9, x FOR PEER REVIEW 17 of 21
activity. In addition, although, the presence of UA and OA in the HE extract could justify the higher
anti-inflammatory activity of the HE extract, compared to the AD extract (Figure 4), the amount of
UA and OA at 50 µg/mL of the HE extract was about 0.1 µM (~50 ng/mL), which is very low.
However, Figure 4A and 4B show that, at each tested concentration, the HE extract is about two-fold
more potent than the AD extract, which corresponds to the ratio of the total phenolic compounds
between the HE and AD extracts (Table 2). Therefore, it can be suggested that other compounds that
are present in higher levels in the HE extract may also contribute to the observed potent
anti-inflammatory activity of T. zygis subsp. zygis HE extract [60]. Thus, we may confirm and
conclude that phenolic compounds of T. zygis subsp. zygis have high anti-inflammatory potential.
Figure 4. Anti-inflammatory activity of Thymus zygis subsp. zygis extracts. (A) Inhibition of nitric
oxide (NO) release by LPS-stimulated RAW 264.7 cells induced by aqueous decoction (AD; left bars,
red) and by hydroethanolic (HE; right bars, blue) extracts, expressed as percentage of control (see
methods for details). (B) Effect of AD (red bars) and HE (blue bars) extracts on RAW 264.7 cells
viability (see methods for details). Results are expressed as mean ± SD (n = 4 independent assays).
Concerning the phenolic composition of these extracts and related contribution to the verified
anti-inflammatory effect, rosmarinic acid has been demonstrated to produce an anti-inflammatory
effect both in in vitro and in vivo experimental models, and by modulating several mechanisms [40].
In RAW 264.7 cells, rosmarinic acid was shown to inhibit inducible nitric oxide synthase (iNOS)
activity [61], resulting in lower NO released levels. The reduction of NO release induced by
rosmarinic acid by LPS-stimulated RAW 264.7 cells was also reported by Martins-Gomes et al. [27].
Recently, other Thymus species, Thymus algeriensis, was reported to have a high content in salvianolic
acid K, together with rosmarinic acid and luteolin glucuronide, which was correlated with the
reported anti-inflammatory activity [62]. The role of the individual phenolic compounds of extracts,
such as the salvianolic acid K and I, in modulating specific cellular pathways involved in
inflammation and proliferation are worth of further study aiming at the discovery of novel
pharmacological relevant molecules.
4. Conclusions
To the best of our knowledge, this is the first work describing the detailed phytochemical
composition of T. zygis subsp. zygis, a Thymus species endemic of Portugal. When compared to other
Thymus species, it contains a higher content of luteolin-(?)-O-hexoside, a polyphenol present in other
Thymus species in lower amounts. Furthermore, its AD extract presented high amounts of luteolin
derivatives including the luteolin-(?)-O-hexoside and luteolin-(?)-O-hexuronide. The amount of total
phenolic compounds of the Thymus species analyzed in this study is comparable to the total phenolic
contents of commercial Thymus species, namely T. vulgaris and T. citriodorus. Additionally, T. zygis
subsp. zygis presented a high antioxidant activity against the ABTS radical and OH radical when
compared to other Thymus species. The AD extract of T. zygis subsp. zygis showed low
Figure 4.
Anti-inflammatory activity of Thymus zygis subsp. zygis extracts. (
A
) Inhibition of nitric oxide
(NO) release by LPS-stimulated RAW 264.7 cells induced by aqueous decoction (AD; left bars, red)
and by hydroethanolic (HE; right bars, blue) extracts, expressed as percentage of control (see methods
for details). (
B
) Effect of AD (red bars) and HE (blue bars) extracts on RAW 264.7 cells viability (see
methods for details). Results are expressed as mean ±SD (n =4 independent assays).
4. Conclusions
To the best of our knowledge, this is the first work describing the detailed phytochemical
composition of T. zygis subsp. zygis, a Thymus species endemic of Portugal. When compared to other
Thymus species, it contains a higher content of luteolin-(?)-O-hexoside, a polyphenol present in other
Thymus species in lower amounts. Furthermore, its AD extract presented high amounts of luteolin
derivatives including the luteolin-(?)-O-hexoside and luteolin-(?)-O-hexuronide. The amount of total
phenolic compounds of the Thymus species analyzed in this study is comparable to the total phenolic
contents of commercial Thymus species, namely T. vulgaris and T. citriodorus. Additionally, T. zygis subsp.
zygis presented a high antioxidant activity against the ABTS radical and OH radical when compared to
other Thymus species. The AD extract of T. zygis subsp. zygis showed low anti-proliferative/cytotoxic
activity, but HE extracts exhibited high anti-proliferative activity. Additionally, both extracts showed
high anti-inflammatory activity, at low concentrations, because they were able to reduce the NO release
by LPS-stimulated RAW 264.7 cells.
T. zygis subsp. zygis has thus a great potential to be used as a functional food, for example as
decoction or herbal tea or as condiment. Furthermore, due to the biological activities presented by the
phenolic compounds, especially in the HE extract, it can also be a source of bioactive ingredients with
antioxidant, anti-proliferative, and anti-inflammatory properties.
Author Contributions:
Conceptualization and experimental design: A.M.S. and F.M.N.; methodology: extraction
procedures, confirmation of chemical analysis and antioxidant activity assays (C.M.-G., F.M.N. and A.M.S.);
performed the HPLC-MS/MS assays (J.S. and M.B.), the HPLC assays (C.M.-G. and F.M.N.) and HPLC
analysis (F.M.N., C.M.-G., J.S., and M.B.); performed the cell viability assays (A.M.S. and C.M.-G.); performed
anti-inflammatory activity assays (A.M.S. and E.B.S.); performed data analysis of cell assays (A.M.S., E.B.S., and
C.M.-G.). Formal data and statistical analysis and data curation: A.M.S., F.M.N., J.A.S., J.S., C.M.-G., M.B., and
E.B.S. Manuscript writing—original draft preparation (A.M.S., F.M.N. and C.M.-G). All authors have read and
corrected/contributed to the final manuscript. Resources and funding acquisition: A.M.S., F.M.N., M.B., and E.B.S.
All authors have read and agreed to the published version of the manuscript.
Funding:
This work was supported by the INTERACT project–“Integrative Research in Environment, Agro-Chains
and Technology”, no. NORTE-01-0145- FEDER-000017, in its line of research entitled ISAC, co-financed by the
European Regional Development Fund (ERDF) through NORTE 2020 (North Regional Operational Program
2014/2020). By funds from the Portuguese Science and Technology Foundation, Ministry of Science and Education
(FCT/MEC) through national funds, under the projects UIDB/04033/2020 (CITAB), UIDB/00616/2020 (CQ-VR) and
UIDB/04469/2020 (CEB). FCT is also acknowledged for the grant to C.M.G. (SFRH/BD/145855/2019).
Acknowledgments:
The authors would like to thank ERVITAL
®
(Plantas Arom
á
ticas e Medicinais, Lda; Mezio,
Portugal) for providing the plants; the Botanical Garden of UTAD for the help with botanical identification, and,
Meriem Taghouti, for collecting the plants and assisting in first plant extractions.
Antioxidants 2020,9, 482 17 of 20
Conflicts of Interest: The authors declare no conflict of interest.
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