Journal of Ethnopharmacology 252 (2020) 112475

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Anti-inflammatory and anticoagulant effects of polyphenol-rich extracts T

from Thymus atlanticus: An in vitro and in vivo study
Tarik Khouyaa,∗, Mhamed Ramchouna,b,c, Souliman Amranic, Hicham Harnafic, Mustapha Rouisd,
Dominique Couchied, Thomas Simmete, Chakib Alema
a
Biochemistry and Natural Substances Team, Department of Biology, Faculty of Sciences & Techniques, University Moulay Ismail, 52000, Errachidia, Morocco
b
Laboratory of Biotechnology & Sustainable Development of Natural Resources, Polydisciplinary Faculty, 23000, Beni Mellal, Morocco
c
Laboratory of Biochemistry and Biotechnologies, Department of Biology, Faculty of Sciences, University Mohamed I, 60 000, Oujda, Morocco
d
Biological Adaptation and Ageing (B2A), CNRS UMR-8256/INSERM ERL U-1164, University Pierre et Marie Curie, Paris, France
e
Ulm University, Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm, Germany

A R T I C LE I N FO A B S T R A C T

Keywords: Ethnopharmacological evidence: Thymus atlanticus (TA) is used in traditional medicine in Morocco to treat chronic
Thymus atlanticus inflammatory diseases, after local and oral treatment.
Rosmarinic acid Aim of study: This study aimed to investigate the in vitro and in vivo anti-inflammatory and anticoagulant ac-
Chicoric acid tivities of an aqueous extract (AE) and polyphenol fraction (PF) derived from TA.
MCP-1
Materials and methods: The effect of AE and PF on monocyte chemoattractant protein-1 (MCP-1) production by
Atherosclerosis
naïve and LPS-stimulated peritoneal macrophages isolated from C57Bl/6 mice was assessed by ELISA assay. The
Macrophage
effect of chronic administration of the extracts at three different doses by oral rout for 2 weeks on blood coa-
Chemical compounds studied in this article: gulation and inflammation induced by carrageenan in Wistar rats was evaluated. In addition, the in vitro an-
Aspirin ticoagulant effect was tested on blood plasma collected from healthy rats using the activated partial thrombo-
Chicoric acid plastin time (APTT), prothrombin time (PT) and thrombin time (TT) tests. The acute toxicity of AE was
Heparin investigated. Phytochemical analysis was carried out by HPLC.
Rosmarinic acid Results: Analysis by HPLC indicated rosmarinic acid as the main phenolic acid in TA extracts. Compared to
control macrophages, MCP-1 level was lower in medium supplemented with AE at 50 and 500 μg/mL and PF at
500 μg/mL, but higher in medium with PF at 50 μg/mL. Rosmarinic and chicoric acids, served as controls,
significantly decreased MCP-1 production. Chronic oral administration of TA extracts prevented inflammation
induced by carrageenan and induced a significant prolongation of blood coagulation time, in a dose dependant
manner, in Wistar rats. The results of the in vitro assay showed that the coagulation time was significantly
prolonged in plasma incubated with extracts in APTT, PT and TT tests. Lethal dose 50 of AE in mice was
27.90 ± 1.19 g/kg.
Conclusion: This study indicated TA as an herb with anti-inflammatory and anticoagulant proprieties and sup-
ports the traditional use of this plant for the treatment of inflammatory diseases.

1. Introduction factors and molecular pathways including oxidative stress, in-

flammatory responses, activated factors of coagulation, and elevated
Atherosclerosis (AS) is the primary cause of several cardiovascular levels of atherogenic lipids (Tapia-Vieyra et al., 2017). Moreover, oxi-
diseases, which are the leading killer worldwide (World Health dative stress promotes the formation of atherogenic particles specifi-
Organization, 2016). AS is a complex process that involves several cally oxidised low-density lipoprotein (ox-LDL) (Kattoor et al., 2018).

Abbreviations: AE, aqueous extract; APTT, activated partial thromboplastin time; AS, atherosclerosis; HPLC, high performance liquid chromatography; LDL, low-
density lipoprotein; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; Ox-LDL, oxidised-LDL; PF, polyphenol fraction; PT, prothrombin time; TA,
Thymus atlanticus; TT, thrombin time
∗
Corresponding author.
E-mail addresses: tarikkhouya@yahoo.com (T. Khouya), ramchoun_10@yahoo.fr (M. Ramchoun), amrani137@yahoo.fr (S. Amrani),
hhicham02@gmail.com (H. Harnafi), mustapha.rouis@upmc.fr (M. Rouis), dominique.couchie@upmc.fr (D. Couchie), thomas.simmet@uni-ulm.de (T. Simmet),
alem04@yahoo.fr (C. Alem).

https://doi.org/10.1016/j.jep.2019.112475
Received 16 April 2019; Received in revised form 26 November 2019; Accepted 9 December 2019
Available online 13 December 2019
0378-8741/ © 2019 Elsevier B.V. All rights reserved.
T. Khouya, et al.
The ox-LDL particles can stimulate immune cells to release chemokines,
which function as chemoattractants that guide the migration and re-
cruitment of immune cells (Balzan and Lubrano, 2018). The monocyte
chemoattractant protein-1 (MCP-1, also known as CCL2), is one of the
key chemokines that play an important role during AS development
(Gregg et al., 2018). MCP-1 has the ability to attract a variety of in-
flammatory cells, especially monocytes, which can transform into
macrophages. The macrophages are the primary cells that interact with
LDL and ox-LDL particles resulting in foam cells, which are the main
cells component of atherosclerotic plaque (Gregg et al., 2018). MCP-1
also initiates the coagulation cascade and activates several serine pro-
teases especially thrombin, which orchestrates proatherogenic events
leading to thrombus and, therefore, to the destabilization of athero-
sclerotic plaque (Bianconi et al., 2018; Jaberi et al., 2018).
Current drugs for AS, such as statins, reduce the risk of cardiovas-
cular events, but are associated with various symptoms including
muscle symptoms, diabetes mellitus, and central nervous system com-
plaints (Thompson et al., 2016). On the other hand, a number of epi-
demiological studies have established an inverse association between
habitual polyphenols intake and incidence of AS (Mendonça et al.,
2019). It appears from experimental studies that phenolic compounds
affect different pathways and risk factors implicated in AS development
(Alagawany et al., 2017). In this regard, our group investigated the
possible effects of species of the Thymus genus, which are widely known
for its high content of polyphenols, on the factors implicated in AS
(Khouya et al., 2015, 2019). Interestingly, it was found that Thymus
atlanticus (TA) reduces LDL accumulation and ameliorates lipid profile
in hyperlipidaemic animal, suggesting a possible beneficial effect on AS
(Ramchoun et al., 2012). TA is an endemic plant in Morocco, the de-
coction of its leaves is traditionally used in the treatment of in-
flammatory diseases after oral and local application (Bellakhdar et al.,
1991). Thus, this study aimed to evaluate how two polyphenol-rich
extracts from TA affect MCP-1 production in macrophages, and in-
flammation and blood coagulation in animal model.
2. Materials and methods
2.1. Reagents and drugs
The APTT (Neoplastin Cl Plus 2, cat. no. 00374), PT (C.K Prest 2,
cat. no. 00598) and TT (STA-Thrombin 2, cat. no. 00610) reagents were
purchased from Diagnostica Stago (Paris, France). ELISA was performed
using a Mouse MCP-1 ELISA Kit MAXTM Deluxe Set (cat. no. 437808)
supplied by Biolegend (Saint-Quentin, France). All other chemical
compounds were purchased from Sigma Aldrich (Verpillière, France).
2.2. Plant material
The leaves of Thymus atlanticus (Ball) Roussine (synonym: Thymus
dreatensis Batt, local name: Ziitra or Azukni, English name: thyme) were
collected from the Tafilalte mountain area in Avril 2017 (32° 15′ N, 5°
25’ E, 1995–2067 m). The plant was identified by Dr. ibn Tatou. A
voucher specimen of this plant has been deposited in the herbarium of
the scientific institute at the University of Mohamed V, Rabat, Morocco
(No. RAB 77496). The name of plant has been checked with http://
www.theplantlist.org/tpl1.1/search?q=thymus+atlanticus.
2.3. Polyphenol-rich extracts from TA
The aqueous extract (AE) was prepared in the same manner as
traditionally used by patients, with some modifications (Khouya et al.,
2019). The polyphenol fraction (PF) was prepared according to a pre-
viously described procedure (Jordán et al., 2009). Both extractions
were repeated three times and the yields of AE and PF were
14.10 ± 1.20% and 8.53 ± 0.50% (w/w), respectively.
The determination of phenolic compounds profile of these extracts
2
Journal of Ethnopharmacology 252 (2020) 112475
was performed using HPLC method (Ramchoun et al., 2012). The total
phenolic content was estimated using Folin-Ciocalteau reagent (Khouya
et al., 2015) and the total flavonoid content was determined using
aluminium chloride method (Ramchoun et al., 2012).
2.4. Animals
C57Bl/6 mice were obtained from the laboratory mice of the
University of Pierre and Marie Curie, Paris, France. Rats and mice were
obtained from the department of biology, faculty of sciences and
techniques of the University Moulay Ismail, Errachidia, Morocco. This
study was carried out in strict accordance with the recommendations in
the Guide for the Care and Use of Laboratory Animals of the European
Union “European directive 86/609/EEC” and of the National Institutes
of Health. The protocol was approved by the Committee on the Ethics of
Animal Experiments of the University of Pierre et Marie Curie, Paris 6,
France (Permit Number: A751301). All surgery was performed under
sodium pentobarbital anaesthesia, and all efforts were made to mini-
mize suffering. Also, this study followed principles in the Declaration of
Helsinki (http://www.wma.net/en/policy/b3.html).
2.5. In vivo study
Male Wistar rats were divided into eight groups of ten animals of
each (n = 10). AE or PF at a dose of 50, 100 or 150 mg/kg/day, aspirin
10 mg/kg/day (positive control) or distilled water (negative control)
were administered orally for 2 weeks. All animals had ad libitum access
to food and water. After 2 weeks, blood was collected via the retro-
orbital sinus, plasma was prepared and the clotting time was measured
by APTT, PT, and TT tests using Diagnostica Stago reagents according
to the manufacturer's instructions. Clotting time was measured using a
model Start 4 coagulometer (Diagnostica, Paris, France) and the results
were expressed in seconds.
Anti-inflammatory effect of chronic extracts administration was
evaluated using carrageenan-induced oedema model. Inflammation was
provoked in all rats with an intraplantar injection of carrageenan
(1.5%) in hind paw. Volume of oedema was measured hourly for 24 h
using a plythesmometre 7140 (Ugo Basile, Gemonio, Italy), and the
percentage of oedema volume and the percentage of oedema inhibition
were calculated (Khouya et al., 2019). Groups treated with distilled
water and aspirin were used as negative control and positive control,
respectively.
2.6. In vitro anticoagulant activity
The in vitro effect of both extracts on APTT, PT and TT was eval-
uated using commercial kits (Diagnostica Stago, France) according to
the manufacturer's instructions with minor modification. Briefly,
plasma prepared from blood of healthy rats (50 μL) was incubated with
10 μL of extract samples, heparin (positive control) or PBS (negative
control) for 5 min before addition of appropriate reagents. Various
concentrations of each extract were tested. Final concentrations of plant
extract in clotting mixture were: 10, 20, 40, 80 and 160 μg/mL of the
clotting mixture. Heparin (final concentration: 1 μg/mL) was used as a
positive control. Results were expressed in seconds (s).
2.7. Inhibition of MCP-1 production
2.7.1. Isolation of mouse peritoneal macrophages
Macrophages were isolated from the peritoneal cavity of female
C57Bl /6 mice. A volume of 2 mL of 4% thioglycolate was injected into
the peritoneal cavity of mice to induce inflammation. After 4 days of
thioglycolate injection, the mice were sacrificed by intra-cardiac in-
jection of lethal dose of pentobarbital. The peritoneal cavity of each
mouse was gently washed with 2 mL of PBS to collect peritoneal cells
suspension. The cell suspension was centrifuged at 100 g for 5 min at
T. Khouya, et al.
Fig. 1. HPLC chromatogram of AE (A) and PF (B). For peak identification at 280 nm, see Table 1.
4 °C to form a cell pellet, which was then resuspended in RPMI-1640
culture medium containing penicillin (100 U/mL) and streptomycin
(100 μg/mL). The cells were counted and seeded in 96-well plates at a
density of 0.5 × 10 6 cells per well. The cells were incubated at 37 °C
under a humidified atmosphere of 5% CO2 for the time necessary to
macrophages adhesion (24 h). The plates were washed three times with
PBS and the adhered cells (macrophages) were incubated overnight in a
medium supplemented with foetal calf serum (FCS) (10%). Next day,
the macrophages were washed three times with PBS and cultured in the
FCS-supplemented medium for 4 days.
2.7.2. Macrophages treatment and ELISA assay
A part of isolated macrophages (naïve macrophages) were stimu-
lated with lipopolysaccharide (LPS, 10 ng/mL, incubation for 2 h). Both
naïve and LPS-activated macrophages were treated with AE, PF, ros-
marinic acid, or chicoric acid at a dose of 50 and 500 μg/mL for 24 h.
After 24 h of treatment, different media were collected and centrifuged
at 700 g, 4 °C for 15 min. The supernatants were recuperated to de-
terminate the amounts of MCP-1 produced by the macrophages. The
MCP-1 amounts were measured by an ELISA assay using a Mouse MCP-
1 ELISA Kit (Biolegend, France). Then, the supernatants were treated
with an anti-MCP-1 capture antibody at 100 μL/well and the media
were maintained overnight at 4 °C. The next day, the media were
3
Journal of Ethnopharmacology 252 (2020) 112475
washed with a wash buffer solution (Tween-20 at 0.05% in PBS) and
incubated for 1 h in presence of a blocking buffer (200 μL/well) at room
temperature, under slow stirring (500 rpm). After washing, 100 μL of an
anti-MCP-1 detection antibody was added to each well and the plates
were incubated at room temperature under stirring for 1 h. The excess
of the antibody was removed by washing, then 100 μL of avidin-HRP
was added into each well and the plates were incubated for 30 min at
room temperature under stirring. The plates were washed five times
1 min of each, and maintained in dark in presence of a peroxidase
substrate (100 μL/well) for 15 min at room temperature. A blue colour
was appeared due to oxidation of substrate. This colorimetric reaction
was stopped by adding 100 μL of a stop solution per each well. The
absorbance of a yellow colour, which appeared after the stopping re-
action, was measured at 450 nm and 570 nm after 15 min of the
stopping reaction. The optical density at 570 nm was subtracted from
that measured at 450 nm.
2.8. Acute toxicity
The acute toxicity study was conducted using the OECD
(Organization for Economic Cooperation and Development)—Guideline
425 (Oecd, 2008). Female albino mice were divided into six groups of
ten mice each. Control group was orally administered distilled water,
T. Khouya, et al. Journal of Ethnopharmacology 252 (2020) 112475

extract-treated groups were orally received AE at 10, 15, 20, 25 and Table 2
50 g/kg BW. All groups were observed for 24 h to determine the lethal Total phenols and flavonoids in AE and PF.
dose 50 (LD50). LD50 was calculated using Trevan's and Kärber's method Sample Total polyphenols (mg gallic Total flavonoids (mg
described by Saganuwan (2017). Control and groups treated with AE at acid/g DE) quercetin/g DE)
20 and 50 g/kg BW were observed for changes in body weight, clinical
Aqueous extract 432.00 ± 11.05 130.91 ± 9.12
and behavioural changes, and adverse reactions, for 2 weeks (Oecd,
Polyphenol 556.85 ± 14.18 179.63 ± 10.00
2008). fraction

2.9. Statistical analysis DE: dry extract.

Results are presented as mean ± standard error (SD) and analysed paw oedema. In control group, the oedema volume progressively rose
by one-way ANOVA or multi-way ANOVA followed by Tukey's or and reached its peak after 5 h of carrageenan injection (augmentation
Dennett's test using a statistical software program Stat View (SAS 30%).
Institute inc., Version 4.0). A p-value of less than 0.05 was considered to In aspirin-treated group (10 mg/kg/day), oedema formation was
denote statistical significance. significantly (p < 0.001) inhibited by 70% after 5 h. Chronic admin-
istration of extracts prevented oedema formation in a dose-dependent
manner (Fig. 2A-B). EA and PF at 50 mg/kg/day exhibited an protective
3. Results
effect similar to that of aspirin (p > 0.05).
3.1. HPLC analysis
3.4. In vitro anticoagulant activity
The phenolic composition of AE and PF was analysed by HPLC
(Fig. 1 and Table 1). The pre-incubation of plasma with AE and PF result in prolongation
The results indicated that rosmarinic acid was the main compound of the clotting time in APTT, PT and TT tests (Table 4).
in both extracts, its content in AE and PF was 4.87 ± 0.33 and At 160 μg of extract per mL of the clotting mixture, both extracts
9.77 ± 0.07 mg/g, respectively. Other phenolic acids were detected in completely inhibited blood coagulation in all tests. AE and PF presented
both extracts including apigenin-7-glucoside, hyperoside and caffeic a significant (p < 0.001) inhibitory effect even at 10 μg/mL. Heparin
acid. Quercetin was detected only in AE (1.15 ± 0.02 mg/g). (1 μg/mL), served as positive control, significantly (p < 0.001) pro-
longed the clotting time compared to negative control.
3.2. Polyphenol and flavonoid contents
3.5. In vitro MCP-1 production of macrophages
The content of total polyphenols in AE and PF was 432 ± 11.05
and 556.85 ± 14.18 mg equivalent gallic acid/g dry extract (Table 2). LPS stimulation increased MCP-1 production by macrophages
The flavonoid content was 130.91 ± 9.12 and 179.63 ± 10.00 mg (Fig. 3). MCP-1 amount produced by naïve and LBS-stimulated mac-
equivalent quercetin/g of dry extract in AE and PF, respectively rophages was 5.53 ± 0.01 and 13.23 ± 1.03 pg/mL (p < 0.001),
(Table 2). respectively.
As shown in Fig. 3A, the AE (50 μg/mL) significantly (p < 0.001)
decreased MCP-1 production in both naïve and LPS-stimulated macro-
3.3. In vivo anticoagulant and anti-inflammatory effects
phages. AE at 500 μg/mL reduced MCP-1 production by LPS-stimulated
macrophages only (Fig. 3A). PF at 500 μg/mL inhibited significantly
As shown in Table 3, the clotting time was significantly (p < 0.01)
(p < 0.001) the MCP-1 production by LPS-stimulated macrophages but
prolonged in a dose dependant manner in rat treated with AE and PF
at low dose (50 μg/mL) it could markedly increase MCP-1 production
when compared with control.
by both naïve and LPS-stimulated macrophages (Fig. 3B). Chicoric and
AE and PF at a dose of 100 mg/kg/day showed an effect on clotting
rosmarinic acids, served as controls, significantly (p < 0.001) in-
time similar to that observed in the group treated with aspirin (10 mg/
hibited MCP-1 production by both types of macrophages, in a con-
kg/day (p > 0.05). Carrageenan-induced paw oedema model was used
centration dependent manner (Fig. 3C-D).
for evaluating of anti-inflammatory effect of chronic administration of
AE and PF (Fig. 2). Results showed that carrageenan injection induces a
3.6. Acute oral toxicity of AE
Table 1
HPLC quantitative analysis of identified polyphenols in AE and PF. The LD50 calculated using Trevan's and Kärber's method was
27.90 ± 1.19 g/kg. Groups that were treated with 20 and 50 g/kg BW
Extract Peak Identified Content (mg/g % in extract
compounds extract)
were observed for 2 weeks for body weight changes (Fig. 4) and other
signs of toxicity. AE at 20 g/kg showed no sign of toxicity and no
Aqueous extract 1 Caffeic acid 0.23 ± 0.03 1.98 change in body weigh during 14 days. However, AE at 50 g/kg induce a
2 Rutin 3.01 ± 0.08 25.97 significant (p ˂ 0.05) reduction in body weigh on the fourth and third
3 Hyperoside 1.15 ± 0.06 9.92
4 Apigenin-7-O- 1.18 ± 0.04 10.18
day after administration. This change disappeared on the fifth day and
glucoside has not appeared since (14 days) (Fig. 4). Salivation (33%, occurrence:
5 Rosmarinic acid 4.87 ± 0.33 42.02 0–3 h) and increased respiration rate (33%, occurrence: 0–2 h) were
6 Quercetin 1.15 ± 0.02 9.92 observed in group treated with AE at 50 g/kg. No sing of ptosis, re-
duction of locomotor activity and tremors were noted.
Polyphenol 1 Caffeic acid 1.43 ± 0.06 9.38
fraction 2 Rutin 0.63 ± 0.01 4.13
3 Hyperoside 1.18 ± 0.03 7.74 4. Discussion
4 Apigenin-7-O- 2.23 ± 0.03 14.63
glucoside
5 Rosmarinic acid 9.77 ± 0.07 64.11 This study was made based on traditional use of TA as anti-in-
flammatory remedy and on the previous findings showing TA as an
Results are expressed as the mean ± SD, (n = 3). hypolipidaemic plant (Ramchoun et al., 2012). Thus, this plant could

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T. Khouya, et al. Journal of Ethnopharmacology 252 (2020) 112475

Table 3
Effect of chronic treatment with AE and PF on blood coagulation in rats.
Test Control (DW) Aspirin (10 mg/kg) Aqueous extract Polyphenol fraction

50 mg/kg 100 mg/kg 150 mg/kg 50 mg/kg 100 mg/kg 150 mg/kg

APTT (s) 18.39 ± 2.37 30.00 ± 2.09** 19.17 ± 4.12 28.12 ± 2.56** 31.07 ± 3.56** 26.58 ± 2.56** 29.53 ± 2.22** 34.37 ± 3.56**
PT (s) 16.13 ± 1.35 27.15 ± 1.43** 19.01 ± 3.24* 22.13 ± 0.58** 25.08 ± 2.16** 20.59 ± 1.53** 21.54 ± 1.45** 28.38 ± 3.35**
TT (s) 21.41 ± 1.87 26.14 ± 2.06** 23.14 ± 2.24* 26.08 ± 2.16** 29.13 ± 3.09** 24.54 ± 1.48** 27.49 ± 2.16** 32.33 ± 1.16**

Rats are orally treated for 2 weeks with distilled water (Control), aspirin (10 mg/kg/day), and aqueous extract or polyphenol fraction at 50, 100, 150 mg/kg/day.
Results were expressed as mean ± SD and analysed by ANOVA followed by Tukey's test, n = 10; *p ˂ 0.01, **p ˂ 0.001, vs. Control.

have a possible anti-atherogenic effect primary as a hypolipidaemic
agent. However, further studies are required to determine the influence
of TA on other risk factors for AS. To this end, we investigated how two
extracts from TA affect inflammation and coagulation in vivo and in
vitro.
Carrageenan-induced oedema is a most used model for evaluation of
anti-inflammatory effect of a new drugs or natural extracts (Ialenti
et al., 2018; Khouya et al., 2019). Carrageenan injection induced an
inflammation response characterised by an infiltration of cells espe-
cially macrophages, increase in exudate and elevated production of pro-
inflammatory mediators such as prostaglandins, leukotrienes, and
chemokines (Ialenti et al., 2018). In the present study, the volume of
carrageenan-induced oedema was quantified to determine the anti-in-
flammatory effects of a chronic administration of the TA extracts.
Compared to control, chronic administration of the extracts prevented
oedema formation in a dose-dependent manner. Both extracts at
100 mg/kg/day exhibited an anti-inflammatory effect similar to aspirin
(10 mg/kg/day). Aspirin is a non-steroid anti-inflammatory drug that
inhibits the activity of the enzyme called cyclooxygenase (COX), leads
to the formation of prostaglandins responsible for inflammation, swel-
ling, pain and fever. Moreover, carrageenan-induced inflammation oc-
curs on two phases. The first phase is sensible to COX-1 inhibitors while
the second phase to Cox-2 inhibitors (Ialenti et al., 2018). Adminis-
tration of TA extracts prevented inflammation development at both
phases suggesting that they inhibited the activity or expression of both
COX-1 and COX-2. COX and its products play a key role in athero-
sclerotic plaque development. Several studies have demonstrated that
prostaglandins synthesis inhibitors blocked AS progression (Bäck et al.,
2019). Zhang et al. (2019) indicated that aspirin can treat AS through
suppressing NFκB1 expression and inactivation of cAMP signalling
pathway in a mouse model of AS. AS is now considered as the
consequence of a complicated inflammatory process at the different
stages of plaque development (Bäck et al., 2019). Among the several
inflammatory molecules implicated in AS, MCP-1 is found to be a
proatherogenic molecule and its elevated level in plasma are a bio-
marker of AS development and associated with death and athero-
sclerotic events in some chronic diseases (Gregg et al., 2018). Moreover,
MCP-1 attracts circulating monocytes that differentiate into macro-
phages, which are important effector cells of atherosclerotic in-
flammation (Bianconi et al., 2018). Inhibition of MCP-1 and regulating
the inflammatory response in macrophages show beneficial effects in
AS development (Gregg et al., 2018). Thus, MCP-1 and macrophages
have become interesting molecular targets for novel drugs to control
and treat AS and cardiovascular diseases (Binesh et al., 2019). In the
present study, the effect of TA extracts on MCP-1 production was stu-
died using peritoneal macrophages from C57Bl/6 mice. The activation
by LPS polarized naïve macrophages to pro-inflammatory type which
produces a high amount of MCP-1. A treatment with TA extracts for
24 h modulated MCP-1 production by naïve and LPS-stimulated mac-
rophages. AE at low and high dose could significantly decreased MCP-1
production. PF at low dose increased MCP-1 production while at high
dose it decreased the MCP-1 production by both naïve and LPS-stimu-
lated macrophages. The effect of extracts was compared to that of two
polyphenols rosmarinic acid, which is the main compounds in TA, and
chicoric acid which is found to decrease the mRNA expression of several
pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-α,
interleukin (IL)-6, and IL-1β (Lee et al., 2015). Rosmarinic and chicoric
acids dramatically decreased MCP-1 production in both types of mac-
rophages. It appears that the effect of the extracts on MCP-1 production
is due to their phenolic compounds especially rosmarinic acid. How-
ever, further investigations are needed to establish the relationship
between PF dose and its effects on MCP-1 production and to study the

Fig. 2. Effect of AE (A) and PF (B) on carrageenan-induced paw oedema in rat. Animals were treated for 2 weeks with AE and PF from TA at 50, 100 or 150 mg/kg/
day. Aspirin at a dose of 10 mg/kg/day was used as a positive control. Control rats were treated only with distilled water. Results are expressed as the mean ± SD
and analysed with Multivariate analysis of variance (MANOVA) followed by Tukey's post hoc test, n = 10. From 2 h, the values of oedema volume differ significantly
(p < 0.001) between treated groups and control.

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T. Khouya, et al. Journal of Ethnopharmacology 252 (2020) 112475

Table 4
In vitro effect of AE and PF on APTT, PT and TT.
Concentration APTT [s] PT [s] TT [s]
μg/mL
AE PF AE PF AE PF

160 ˃ 600* ˃ 600* ˃ 300* ˃ 300* ˃ 300* > 300*

80 ˃ 600* ˃ 600* 100.33 ± 0.94* ˃ 300* ˃ 300* 155.41 ± 0.06*
50 322.58 ± 2.65* ˃ 600* 21.27 ± 0.44* 19.97 ± 0.25* 119.38 ± 0.35* 115.62 ± 0.06*
40 65.50 ± 2.25* 105.74 ± 1.61* 17.13 ± 0.43* 17.93 ± 1.16* 86.69 ± 0.33* 85.48 ± 0.30*
20 32.22 ± 3.22* 24.14 ± 0.18* 14.13 ± 0.14* 16.63 ± 1.16* 46.02 ± 0.02* 59.81 ± 0.63*
10 24.07 ± 0.25* 20.44 ± 0.01* 13.12 ± 0.22 14.00 ± 1.09 38.30 ± 0.23* 38.70 ± 0.03*
PBS 15.10 ± 0.11 15.10 ± 0.12 13.02 ± 0.28 13.02 ± 0.29 18.44 ± 0.02 18.44 ± 0.02
Heparin 51.68 ± 0.83* 51.68 ± 0.83* 60.03 ± 1.54* 60.03 ± 1.54* 50.78 ± 1.54* 50.78 ± 1.54*

AE: aqueous extract, PF: polyphenol fraction. Concentration is represented as μg of extract in mL of clotting mixture. PBS and heparin (1 μg/mL) were used as
negative and positive controls, respectively. Results are expressed as the mean ± SD and analysed with one-way ANOVA followed by Dennett's post hoc test, n = 6.
*p < 0.001 vs. control.

mechanisms of anti-MCP-1 effect of TA on macrophages. To date, a few
works have examined the effect of plant and polyphenols on the pro-
duction of MCP-1. A recent study indicated MCP-1 as a possible target
for the anti-inflammatory effects of curcumin, a natural polyphenol
isolated from curcuma (Karimian et al., 2017). Curcumin is found to
modulate the activity and production of MCP-1 through influencing
MAPK and NF-kB signalling pathways (Karimian et al., 2017).
Aside from their anti-inflammatory effects, we also investigated the
effect of TA extracts on coagulation system in vitro and in vivo.
Generally, there are two separated pathways of blood coagulation in-
itiation, intrinsic and extrinsic pathways, which converge on common
pathway. The goal of coagulation is to stop the loss blood after vascular
injury. However, under pathologic conditions, inflammatory mediators
and oxidative stress induce an overproduction of coagulation products,
particularly thrombin, which have pleotropic effects leading to the
rupture of atherosclerotic plaque (Jaberi et al., 2018). In addition,
normal and atherosclerotic vascular cells express protease-activated
receptors (PAR) that respond to thrombin resulting in their prolifera-
tion via the activation of a nuclear factor NF-kB and, therefore, in
atherosclerotic complications (Bochenek and Schäfer, 2019). In the
current study, TA extracts acted as oral anticoagulants and caused a
prolongation of APTT, PT and TT suggestion that they inhibited the
activity or production of the factors implicated in intrinsic and extrinsic
pathways, and fibrinogen synthesis. Additionally, TA extracts could

Fig. 3. Effect of AE (A), PF (B), rosmarinic acid (C) and chicoric acid (D) on MCP-1 production by naïve and LPS- stimulated peritoneal macrophages isolated from
C57BL/6 mice. RA: rosmarinic acid, CA: chicoric acid. Naïve (LPS −/−) and LPS-stimulated (LPS +/−) macrophages were treated with two doses of each sample
(50 and 500 μg/mL). MCP-1 amount was measured by ELISA. Data are expressed as mean ± SD and analysed with one-way analysis of variance (ANOVA) followed
by Tukey's post hoc test, n = 3. a: ns: not significant, *p < 0.001 vs. naïve macrophages; b: ns: not significant, *p < 0.001 vs. LPS- stimulated macrophages.

6
T. Khouya, et al.
Fig. 4. Body weight change during 14 days. Female albino mice received dis-
tillate water (Control) or AE at 20 and 50 g/kg BW (n = 10 per group). Data are
expressed as mean ± SD and analysed with One-way ANOVA followed by
Dunnett's post hoc test, n = 10. *p < 0.05, vs. control.
prolong directly the clotting time in vitro by inhibition intrinsic, ex-
trinsic and common pathways. This effect can due to a direct inhibition
of activated factors, which suggests the possible beneficial effect of this
plant on the development and destabilization of atherosclerotic plaque.
Moreover, a number of studies have confirmed the beneficial effect of
coagulation protease inhibitors on the attenuation of AS progression in
animal models (Posma et al., 2019).
Another candidate implicated in AS pathogenesis is oxidative stress,
which results from the imbalance between ROS and antioxidants in
living cells. Endogenous and exogenous antioxidants can neutralize and
suppress different ROS forms and, therefore, can be beneficial agents in
the development of diseases. A recent study (Kartikey et al., 2019) has
established a relationship between dietary total antioxidant capacity
and AS mortality. It concluded that antioxidant-rich diets are beneficial
in reducing risk of AS. To date, only the in vitro antioxidant effects of TA
have been confirmed (Khouya et al., 2015; Ramchoun et al., 2012).
Thymus genus is well known as a rich source of phenolic compounds
and flavonoids. Our results on phytochemical screening are in agree-
ment with previous findings (Khouya et al., 2015; Ramchoun et al.,
2012). We found that AE and PF of TA contain various phenolic com-
pounds with rosmarinic acid as the major compound in both extracts.
Polyphenols are among the most bioactive natural agents in the pre-
vention and the treatement of chronic inflammatory diseases. In clinical
studies, the intake of polyphenols showed an inverse association with
the risk of AS-related diseases (Mendonça et al., 2019). Moreover, a
recent review (Alagawany et al., 2017) indicated that rosmarinic acid
can prevent AS and its associated diseases by reducing pro-in-
flammatory mediators and enzymes, inhibiting recruitment of in-
flammatory cells, and decreasing the production of different forms of
ROS by immune cells. These data can explain the biological activities of
TA extracts, which have a high content in rosmarinic acid.
Administration of AE at 20 and 50 g/kg BW did not cause any severe
signs of toxicity. The oral lethal dose (LD50) of TA is higher than 20 g/
kg, and can be classified as a low toxicity extract according to OECD
(Oecd, 2008).
5. Conclusions
AS is a chronic inflammatory disease which implicates multiple
factors. Our results provide the evidence that TA extracts can affect AS
and inflammatory diseases by modulating MCP-1 production, inhibiting
intrinsic, extrinsic and common pathways of coagulation. TA is a non-
toxic plant that represents a new resource for the development of nat-
ural molecules with anti-inflammatory effects and it is suggested to
purify from this plant the molecules to test, in vivo and in vitro, on
biological models more pertinent for a live application and most ad-
vanced for the prevention and the treatment of AS and inflammatory
7
Journal of Ethnopharmacology 252 (2020) 112475
diseases.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Author contributions
This study was conceived by T.K, C.A and S.A. T.K. conducted the in
vivo anti-inflammatory and anticoagulant studies. T.K., M.R. and C.A.
were participated in preparation of plant materiel and extracts, the
determination of quantitative contents and the anticoagulant effect in
vitro. S.A., M.R., and D.C. conducted the study on MCP-1. T.S. con-
ducted HPLC technique. T.K., M.R., S.A., H.H. and C.A. were con-
tributed to the writing and discussion of the paper.
Declaration of competing interest
None.
Acknowledgments
The authors wish to express their gratitude to Dr. Ibn Tatou for plant
material identification.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jep.2019.112475.
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