Scientific African 11 (2021) e00716

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Scientific African
journal homepage: www.elsevier.com/locate/sciaf

Phytochemical analysis and bioactivity evaluation of

Moroccan Thymus atlanticus (Ball) fractions
Tarik Khouya a,∗, Mhamed Ramchoun a,b,c, Abdelbassat Hmidani a,
Eimad dine Tariq Bouhlali d, Souliman Amrani c, Chakib Alem a
a
Biochemistry and Natural Substances Team, Department of Biology, Faculty of Sciences & Techniques, University Moulay Ismail,
Errachidia 52000, Morocco
b
Laboratory of Biotechnology & Sustainable Development of Natural Resources, Polydisciplinary Faculty, University Sultan Moulay
Slimane, Beni Mellal 23000, Morocco
c
Laboratory of Biochemistry, Department of Biology, Faculty of Sciences, University Mohammed I, Oujda 60000, Morocco
d
National Institute of Agronomic Research, Regional Center of Errachidia, 52000, Morocco

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

Article history: This study aims to evaluate the effect of polar and less polar fractions of Thymus atlanticus
Received 16 September 2020 (T. atlanticus) on inflammation and hyperlipidemia in animal models, and on coagulation
Revised 29 December 2020
and lipid peroxidation in vitro. Polar and less polar fractions of T. atlanticus were analyzed
Accepted 27 January 2021
by HPLC and their antioxidant activities were evaluated using different in vitro methods.
The anti-inflammatory effect was tested using arachidonic acid and xylene-induced inflam-
Keywords: mation tests in animal models. Acute hyperlipidemia was induced in mice by an intraperi-
Dyslipidemia toneal injection of Triton WR-1339. After 24h, plasma was collected to analyze lipid profile.
Arachidonic acid The effect of the fractions and heparin on coagulation of the rabbit blood was explored in
Inflammation vitro by partial thromboplastin time, prothrombin time and thrombin time tests. The re-
Thymus atlanticus sults indicated that rosmarinic acid was the main phenolic acid. All fractions significantly
Flavonoids
protected plasma lipids against oxidation. The ethyl acetate fraction significantly inhibited
Phenolics
inflammation induced by arachidonic and xylene in mice and rat, respectively. The admin-
istration of fractions at a dose of 2 g/kg body weight markedly decreased plasma total
cholesterol, triglycerides and LDL-C but only the aqueous fraction increased the level of
HDL-C in hyperlipidemic mice. The blood coagulation was completely inhibited even at
very low concentration of fractions (280 μg/mL of plasma). T. atlanticus could be a source
of potential lipid-lowering and anti-inflammatory compounds.
© 2021 The Authors. Published by Elsevier B.V. on behalf of African Institute of
Mathematical Sciences / Next Einstein Initiative.
This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/)

Abbreviations: AA, arachidonic acid; ABTS, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid; AF, aqueous fraction; AI, atherogenic index; BHA,
butylated hydroxyanisole; CVDs, cardiovascular diseases; DCMF, dichloromethane fraction; EAF, ethyl acetate fraction; HCM, hyperlipidemic control mice;
HDL-C, high-density lipoprotein cholesterol; HPLC, high performance liquid chromatography; LDL-C, low-density lipoprotein cholesterol; MDA, malondi-
aldehyde; MF, methanol fraction; NCM, normolipidemic control mice; Ox-LDL-C, oxidized-LDL-C; PT, prothrombin time; PTT, partial thromboplastin time;
T. atlanticus, Thymus atlanticus; TBARS, thiobarbituric acid reactive substances; Triton, triton WR-1339; TT, thrombin time.
∗
Corresponding author.
E-mail address: tarikkhouya@yahoo.com (T. Khouya).

https://doi.org/10.1016/j.sciaf.2021.e00716
2468-2276/© 2021 The Authors. Published by Elsevier B.V. on behalf of African Institute of Mathematical Sciences / Next Einstein Initiative. This is an
open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
T. Khouya, M. Ramchoun, A. Hmidani et al. Scientific African 11 (2021) e00716

Introduction

Medicinal plants provide an important source of beneficial compounds for human health. Plants have been used for
thousands of years in traditional medicine for preventing and treating many ailments [1]. However, until now, only a few
number of plants are explored. Plants contain mixtures of chemical compounds that act on the human body to prevent
diseases and to maintain or restore health [2]. The medicinal properties of many plants have been attributed mainly to the
presence of polyphenols and flavonoids [3]. These phytochemicals initially discovered as potent antioxidants are also known
to possess anti-inflammatory, anticancer, analgesic and anticoagulant properties [3].
Recent epidemiological studies have been reported that polyphenols intake is conversely associated with the incidence
of cardiovascular diseases (CVDs) [4]. CVDs are a major cause of death in the world [5]. Unfortunately, the development
of CVDs is associated with many risk factors and involves several physiological mechanisms including inflammation and
coagulation as well as metabolism of lipids [6,7]. However, the current strategy for controlling CVDs is based only on the
use of lipid-lowering drugs such as statins and fibrates. Although effective, statins and fibrates are associated with many
side effects [8,9]. Therefore, scientists are interested to find effective drugs with anti-hyperlipidemic, anti-inflammatory and
anticoagulant properties for the control of CVDs [10,11]. Polyphenol-rich extracts derived from plants may provide a feasible
alternative as an adjunct therapy to statins and fibrates in CVDs [12]. Unlike medications, which have one or few active
ingredients, a single plant contains diverse secondary metabolites and can show pleiotropic effects [13].
Thymus atlanticus (T. atlanticus) is an endemic herb in Morocco [14]. Generally, Thymus species have been used in Mo-
roccan traditional medicine to treat all diseases such as gastro-intestinal disorders and for its antiseptic properties [1]. It has
been reported that several of these Thymus species have been used for their anti-inflammatory benefits to manage bron-
chitis, whooping cough and rheumatism [14]. The assessment of lipid-lowering effect of some endemic Thymus varieties of
Morocco demonstrated that T. atlanticus exhibited an effective anti-hyperlipidemic effect, a rarely described effect in Thy-
mus species [17]. T. atlanticus, commonly known as thyme, grows in Mediterranean regions and is widely spread in Morocco
especially in the High Atlas Mountains at altitudes of 190 0–20 0 0 m [14]. Thymus genus comprises more than 300 species,
most of which remain scientifically unexplored [15]. Thus, this study focused on the evaluation of biological activities of
fractions derived from Moroccan T. atlanticus.
Aqueous extract of T. atlanticus has been found to have potent antioxidant activity in vitro and to inhibit effectively
inflammation provoked by carrageenan and croton oil in animal models [16,17]. The first aim of the present study was to
confirm the biological activities of T. atlanticus and to justify its traditional use by studying its effects on some mechanisms
contributing to the pathogenesis of various diseases such as heart disease.
Previous phytochemical analysis of the aqueous extract of T. atlanticus revealed the presence of three phenolic compounds
only: rosmarinic acid, caffeic acid and quercetin [18]. However, the chemical composition of T. atlanticus is still incomplete. In
this regard, the present study focused on the phenolic composition of polar and less polar fractions derived from T. atlanticus
and their anti-inflammatory, antioxidant and anti-hyperlipidemic effects with a view of screening the active fraction (s).

Materials and methods

Materials and chemicals

Plythesmometer 7140 was purchased from UGO Basile (Salon-de-Provence, France). STart 4 Hemostasis Analyzer was
purchased from Diagnostica Stago (Asnières-sur-Seine, France). PT (C.K Prest 2, cat. no. 00598), PTT (Neoplastin Cl Plus 2,
cat. no. 00374) and TT (STA-Thrombin 2, cat. no. 00610) reagents were obtained from Diagnostica Stago (Paris, France). All
other chemical compounds were purchased from Sigma Aldrich (Verpillière, France).

Plant material and extraction

The aerial parts of T. atlanticus (Ball) Roussine were collected from the Tafilalet Mountain area (32° 15’ N, 5° 25’ E,
1995 m), in Errachidia Province in Morocco, in April 2016. T. atlanticus has been identified by Dr. Ibn Tatou and a voucher
specimen has been deposited in the herbarium of the scientific institute at the University of Mohammed V. Rabat, Morocco
(No: RAB 77496).
To prepare different fractions, about 60 g of dried powder of the aerial parts was first degreased with n-hexane (C6 H14 )
in a Soxhlet extractor. Then, the obtained marc was sequentially exhausted completely with different solvents (380 mL) with
increasing polarity: dichloromethane (CHCl3 ; polarity index (P’) = 4.1), ethyl acetate (C4 H8 O2 ; P’ = 4.4), methanol (CH3 OH;
P’ = 5.1), and at the end, the marc was infused in distilled water (H2 O; P’ = 10.2). The marc was air-dried before each
extraction with the appropriate solvent for 10-12 h. The extraction yields, following the removal of solvent using a rotary
evaporator, were 6%, 6.5%, 10% and 4% for dichloromethane fraction (DCMF), ethyl acetate fraction (EAF), methanol fraction
(MF) and aqueous fraction (AF), respectively.

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Total polyphenol, flavonoid and tannin contents

Total polyphenol content

Total polyphenol contents were determined according to the Folin-Ciocalteu colorimetric method [17]. Briefly, 30 μL of
sample (fraction) was mixed with 4.22 mL of distilled water, 250 μL of the Folin-Ciocalteau reagent and 500 μL of 10%
sodium carbonate in a test tube. The mixture was allowed to stand for 30 min and the absorbance was measured using
spectrophotometry at 725 nm. The content of total polyphenols was expressed as mg of caffeic acid equivalent per g dried
residue (DR).

Total flavonoid content

Flavonoid contents in plant fractions were determined using aluminum chloride in a colorimetric method [19]. First, the
aluminum chloride solution was prepared by dissolving 133 mg of AlCl3 in 100 mL of a mixture solvent of methanol-water-
acetic acid (70:25:5, v/v/v). Then, the 1 mL of fraction and 0.5 mL of aluminum chloride solution were mixed in a test
tube, allowed to stand for 30 min and the absorbance was measured at 430 nm. The results were expressed as mg of rutin
equivalent per g DR.

Total tannin content

Total tannin content was determined using the method described by Heimler et al. [19]. Sample (50 μL), 4% methanol
vanillin solution (3 mL) and concentrated hydrochloric acid (1.5 mL) were introduced in a test tube, mixed and the tube
was allowed to stand for 15 min. Then, the absorbance was measured at 500 nm against methanol as a blank. Catechin was
used to prepare a calibration curve and the contents were expressed as mg of catechin equivalent per g of DR.

High performance liquid chromatography (HPLC) analysis

The polyphenols profiling of fractions derived from T. atlanticus were performed according to the procedure of Khouya
et al. [16] using a Reprosil Pur C18 column (250 × 3 mm, 5 μm) equipped with a photodiode array detector with a LC-
20AB model dual pump. The column oven temperature was set at 28°C. The flow rate of elution was maintained at 0.5
mL/min. The acquisition wavelengths were set at 215, 250, and 280 nm. A binary gradient solvent system was used: (A)
methanol/water (20/80) + 0.2% glacial acetic acid and (B) methanol/water (80/20) + 0.2% glacial acetic acid [100% (A) and
0% (B) at 0 min, 50% (A) and 50% (B) during 10 min, 17% (A) and 83% (B) during 20 min, which was changed to 100% (A) and
0% (B) in 5 min (35 min, total time)]. Standard stock solutions (100 μg/mL): caffeic acid, rosmarinic acid, rutin, quercetin,
apigenin, hesperetin, luteolin-7-glucoside and daidzein were used to prepare calibration curves. Before injection, one gram
of extract was dissolved in 25 mL acidified methanol solution (1N HCl/methanol/water, 1/80/19, v/v/v). The compounds in T.
atlanticus fractions were identified based on the retention time of standards and the corresponding UV spectra. The results
were expressed as mg of phenolic compound per g of DR.

Antioxidant activity

ABTS radical scavenging assay

The radical scavenging activity (RSA) was evaluated by 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)
assay according to the method described by Re et al. [20]. To obtain the solution of ABTS radical cations (ABTS+ ), 2.45
mM aqueous solution of potassium persulfate was added to 7 mM aqueous solution of ABTS. The mixture was allowed
to stand overnight in the dark at ambient temperature, and then diluted with distilled water to obtain an absorbance of
0.70 0 ± 0.0 05 at 734 nm. Different concentrations of the sample (15 μL) were introduced in test tubes with 1.5 mL of
the ABTS solution, the tubes were incubated for 5 min at ambient temperature, then the absorbance was measured at 734
nm. Different concentrations of ascorbic acid were used to made the calibration curve. The RSA was expressed as mmol of
ascorbic acid equivalent per g of DR.

β -carotene/linoleic acid assay

The antioxidant activity of the fractions was determined according to the β -carotene bleaching method [21]. Two
milliliters of β -carotene solution (0.2 mg/mL in chloroform), 20 μL linoleic acid and 200 μL Tween 20 were introduced
in a round-bottomed flask and the mixture was then evaporated at 40°C for 10 min to remove the solvent. Then, 100 mL
of distilled water was immediately added. After agitating, 1.5 mL of the resulting emulsion was transferred into test tubes
containing 150 μL of sample (plant fraction) and the absorbance was measured at 470 nm against a blank consisting of an
emulsion without β -carotene. The tubes were placed in a water bath at 50°C and the oxidation of the emulsion was moni-
tored by measuring absorbance at 470 nm using a spectrophotometry. The same procedure was repeated with the synthetic
antioxidant, butylated hydroxyanisole (BHA), served as positive control. The inhibition of the β -carotene bleaching after 2 h
was calculated by the following equation:

Inhibition of bleaching (% ) = ([β − carotene after 2 h]/[β − carotene initial] ) ∗ 100

The IC50 value is the concentration of each sample required to give 50% β -carotene bleaching.

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Malondialdehyde (MDA) assay

Thiobarbituric acid reactive substances (TBARS) method was used to determine the ability of the fractions to protect
plasma lipid against peroxidation induced by copper [17]. Lipid-rich plasma obtained from mice injected with Triton WR-
1339 (Triton) at a dose of 600 mg/kg BW was used as substrate for oxidative process. In the control tube, plasma was
incubated with distilled water only. In the second test, oxidation was inducted with 10 μL of copper sulfate (CuSO4 ; 5H2 O)
solution (0.33 mg/mL). In the test tubes, oxidation was inducted by copper and fractions were added at 25, 50, 10 0 and 20 0
mg/mL. Then, 100 μL of 8.1 % (w/v) sodium dodecyl sulfate (SDS) was added to each assay and the mixtures were stirred
and incubated for 60 min at room temperature. Afterward, the reaction mixture was heated at 95°C for 60 min after the
addition of 250 μL of 20% trichloroacetic acid (pH: 3.5) and 250 μL of 0.8% (w/v) thiobarbituric acid (TBA). After cooling, 1
mL of n-butanol was added and the tubes were centrifuged at 10 0 0 g for 15 min and the absorbance of resulting colored
layer was measured at 532 nm. The amount of TBARS was calculated as mmol MDA equivalent from a calibration curve
made with 1, 1, 3, 3-tetramethoxypropane standard solutions.

Animals

Thirty-six male Wistar rats (150–200 g), thirty-six male Wistar mice (20–30 g), and six albino rabbits (1500- 2500 g) were
obtained from the animal facility of the Department of Biology (Faculty of Sciences, Errachidia, Morocco). Eighty male albino
mice, weighing 24–28 g, were provided from the animal facility of the Department of Biology (Faculty of Sciences, Oujda,
Morocco). All animals were housed in metallic cages (three animals per cage) under controlled temperature (22–24°C), 12h
light/12h dark cycle and relative air humidity 40%–60%. Flake wood shavings were used as bedding materials. The animals
had continuous access to food and water. A standard chow diet, containing 23% proteins, 58% starch, 6% cellulose, 5% lipids,
8.5% minerals and 1.2% vitamins, was obtained from SONABETAIL society (Oujda, Morocco). All experiments were conducted
in accordance with the guidelines regarding the Care and Use of Laboratory Animals, published by the US National Institutes
of Health ((NIH Publications No. 8023, revised 1978) as approved by the Animal Research Committee at the University of
Moulay Ismail and the University of Mohammed I (Oujda, Morocco). Animal experiments were carried out under isoflurane
anesthesia. The mice were euthanized at the end of the experimental period by an intraperitoneal injection of sodium
pentobarbital at 60 mg/kg body weight (BW).

Acute anti-hyperlipidemic effect

To induce acute hyperlipidemia, the animals were intraperitoneally injected with 0.2 g/kg BW of Triton WR-1339 [17].
Male mice were divided into eight groups of ten mice each (n = 10). Normolipidemic control mice (NCM) were not injected
with Triton and received only distilled water by oral rout. Hyperlipidemic control mice (HCM) and hyperlipidemic dimethyl
sulfoxide (DMSO) control mice (HDCM) were injected with Triton and received orally distilled water and DMSO (1%), re-
spectively. The fractions (2 g/kg BW) and fenofibrate (65 mg/kg BW, served as positive control) were orally administered to
mice one hour after Triton injection.
The animals had access only to water throughout the 24h period following treatment. After 24h of treatment, blood
was collected into heparinized tubes from the retro-orbital sinus of mice under slight anesthesia with isoflurane. The blood
samples were immediately centrifuged at 1500 g for 15 min and plasma was used for lipid analysis.
Plasma levels of total cholesterol, triglycerides, LDL-C and HDL-C were determined using commercial reagents (Biosud
Diagnostici S.r.l. Italy) according to the method of Ramchoun et al. [17]. The LDL-C/HDL-C ratio was calculated as the ratio
of plasma LDL-C to HDL-C levels; Atherogenic index (AI) was calculated using the following equation:
AI = (Total cholesterol − HDL − C )/HDL − C

Anti-inflammatory activity

Xylene-induced ear edema

The topical anti-inflammatory activity of the fractions was evaluated as inhibition of the croton oil-induced ear edema
in mice [22]. Male Wistar mice were divided into six groups of six animals each. For the mice in negative control, 25 μL
of xylene was applied on the inner surface of right ear to induce edema, while the left ear was considered as control. In
the positive control, indometacin (400 μg/ear) was mixed with xylene and applied on the iner surface of the right ear. In
test groups, the different fractions (800 μg/ear) dissolved in xylene were topically applied to the right ears of the mice. The
left ear remained untreated. The evolution of ear edema was determined hourly by measuring the thickness with a digital
thickness gauge. The ear edema rate and the inhibition rate of each group were calculated as follows:
Ear edema rate (% ) = 100 ∗ (Tr − Tl /Tl )

Inhibition rate (% ) = 100 ∗ (Ec − Et /Ec )

where Tr is the right ear thickness and Tl is the thickness of the left ear (untreated ear); Ec is the ear edema rate of the
control group and Et is the ear edema rate of the treated group.

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Table 1
Total polyphenol, flavonoid and tannin contents of T. atlanticus fractions.

Fractions Polyphenols (mg equivalent gallic acid/g DR) Flavonoids (mg equivalent rutin/g DR) Tannins (mg equivalent catechin/g DR)

DCMF 61.88 ± 0.68 36.00 ± 2.10 25.75 ± 5.97

EAF 556.88 ± 2.15 28.18 ± 0.87 20.03 ± 0.18
MF 371.25 ± 3.12 56.59 ± 2.08 61.95 ± 1.73
AF 185.63 ± 3.05 31.18 ± 0.47 35.80 ± 5.93

AF: aqueous fraction, DCMF: dichloromethane fraction, DR: dried residue, EAF: Ethyl acetate fraction, MF: methanol fraction. Results are expressed
as the mean ± SD, (n = 3).

Arachidonic acid (AA)-induced paw edema

Rats were divided into six groups of eighty animals each (n = 8). Edema was induced by injecting 100 μL of AA (1
mg/mL of carbonate buffer solution) into the sub-plantar region of the right hind paw [23]. Indomethacin (5 mg/kg, served
as positive control) and the fractions (25 mg/kg) were orally administered to rats 30 min before AA injection. Control rats
were injected with AA and received only distilled water. Paw volume was measured immediately after the injection of
AA and at 30-min intervals for 4h using a plythesmometer (UGO Basile 7140, France). Percentage inhibition of edema was
calculated using following equations:
Edema rate (E ) % = 100 ∗ (V0 − Vt /V0 )

% Inhibition = 100 ∗ (Ec − Et /Ec )

Where V0 is the paw volume before AA injection and Vt is the paw volume after AA injection at ‘t’ min, Ec is the edema
rate of the control group and Et is the edema rate of treated group.
The dose of the fractions and indomethacin was selected based on previous in vivo experiments [16,18].

Clotting assays

Blood was collected into citrated tubes from the lateral marginal ear vein of six healthy rabbits. The blood samples were
immediately centrifuged at 2500 g for 10 min and the plasma was collected. The fractions were diluted with phosphate
buffered saline (pH 7.4) to obtain the following concentrations: 0.3125, 0.625, 1.25, 2.5, 5, 10 and 20 mg/mL. Final concen-
trations of the fractions in clotting mixture were: 8.75, 17.5, 35, 70, 140, 280 and 560 μg/mL.
The anticoagulant effect of the fractions was evaluated using three screening tests: prothrombin time (PT), partial throm-
boplastin time (PTT) and thrombin time (TT). PTT and PT were carried out as previously described [16]. In TT, 50 μL of sam-
ple (fraction) was incubated with 100 μL of plasma at 37°C for 5 min before adding TT-reagent (Diagnostica Stago, France).
Meanwhile, the clotting time was recorded using a coagulometer (Stago, Start 4, France). The results were expressed in sec-
onds. Physiological saline and sodium heparin were served as negative control and positive control, respectively. All clotting
assays were performed in six replicates for each concentration to estimate the mean clotting time.

Statistical analysis

The data were presented as means ± standard deviation and analyzed by analysis of variance (ANOVA) for repeated
measures and then post hoc analysis (Tukey’s test). A P value less than 0.05 was considered statistically significant.

Results

Chemical composition

Table 1 shows the results of total polypenol, flavonoid and tannin contents of different T. atlanticus fractions. The organic
solvents showed different abilities of extracting polyphenols. Ethyl acetate was the best solvent for efficient quantitative
extraction of phenolic acids, followed by methanol, where 556.88 ± 2.15 and 371.25 ± 3.12 mg of gallic acid/g of DR were
obtained, respectively.
Compared to other solvents, dichloromethane had the lowest ability in extracting the phenolic acids. Flavonoids represent
59%, 5%, 15% and 16% of total polyphenols weight in DCMF, EAF, MF and AF, respectively. In addition, methanol was the best
organic solvent for the quantitative extraction of tannins (61.95 ± 1.73 mg of catechin/g DR) (Table 1).
The HPLC chromatograms of the fractions are given in Fig. 1 and the amounts of phenolic compounds detected in the
fractions are shown in Table 2. Various compounds were detected: caffeic acid, rosmarinic acid, quercetin, rutin, hyperoside
and luteolin-7-O-glucoside. Quercetin was detected only in DCMF. Rosmarinic and caffeic acids were found in three fractions
(EAF, MF and AF). Rosmarinic acid was the predominant phenolic acid. Its level was about 4.43 mg/g, 5.09 mg/g and 0.23
mg/g DR in EAF, MF and AF, respectively. EAF and MF were particularly rich in hyperoside and luteolin-7-O-glucoside. These
two fractions were the richest fractions in phenolic acids and flavonoids.

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Fig. 1. HPLC chromatograms of the T. atlanticus fractions.

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Table 2
Quantitative HPLC analysis of polyphenols in T. atlanticus fractions.

Fractions Caffeic acid Rosmarinic acid Quercetin Rutin Hyperoside L-7-O-glu

DCMF - - 0.12 - - -
EAF 2.38 4.43 - 0.42 1.53 3.03
MF 1.94 5.09 - - 0.99 1.76
AF 0.19 0.23 - - - -

Results are expressed as mg of phenolic compound per g of dried residue (n = 1). AF: aque-
ous fraction, DCMF: dichloromethane fraction, EAF: ethyl acetate fraction, L-7- O-glu: luteolin-7-
O-glucoside, MF: methanol fraction. -: absent.

Table 3
Antioxidant activity of the fractions of T. atlanticus evaluated by β -carotene and ABTS assays.

Samples Inhibition of β -carotene bleaching (IC50 mg/mL) ABTS (mmol ascorbic acid/g dried residue)
∗
DCMF 4.30 ± 0.02 2.33 ± 0.21
EAF 0.20 ± 0.08∗ 7.73 ± 0.43
MF 0.20 ± 0.04∗ 8.06 ± 0.06
AF 0.79 ± 0.04∗ 5.39 ± 0.35
BHA 0.09 ± 0.01 -

AF: aqueous fraction, DCMF: dichloromethane fraction, EAF: ethyl acetate fraction, MF: methanol fraction, –:
absent. Results are expressed as the mean ± SD and analyzed with one-way ANOVA followed by Tukey’s post
hoc test, n = 6, ∗ p < 0.05 (vs. BHA).

Fig. 2. Effect of the fractions of T. atlanticus on plasma lipid peroxidation mediated by CuSO2 . AF: aqueous fraction, BHT: butylated hydroxytoluene, Control:
non-oxidized control, EAF: ethyl acetate fraction, MF: methanol fraction, OX: oxidized control. Results are expressed as the mean ± SD and analyzed with
one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05 vs. control.

Antioxidant activity

The antioxidant activity of different fractions was assessed by ABTS, β -carotene and TBARS assays. The ABTS scavenging
activity of the fractions ranged from 2.33 ± 0.21 to 8.06 ± 0.06 mmol of ascorbic acid/g DR (Table 3). MF showed the highest
antioxidant activity based on ABTS method (8.06 ± 0.06 mmol/g DR), while DCMF had the lowest ABTS scavenging activity
(Table 3).
As shown in Table 3, all fractions were able to inhibit β -carotene bleaching. EAF and MF exhibited the highest inhibitory
effect and their IC50 values were 0.20 ± 0.08 and 0.20 ± 0.04 mg/mL, respectively. The results showed also that the lowest
activity was found in DCMF with an IC50 value of 4.30 ± 0.02 mg/mL. All fractions showed lower effect (p < 0.001) than
BHA having an IC50 value of 0.09 ± 0.01 mg/mL. The results of the protective effect of the fractions against plasma lipids
oxidation are given in Fig. 2. Compared to oxidized control, EAF, MF and AF presented relatively high inhibitory effect at 25
mg/kg. No significant difference in the activity between fractions was noted. BHT, which served as a positive control, had
higher inhibitory effect on plasma lipid oxidation than fractions.

Effects of the fractions on acute hyperlipidemia in mice

Comparing to NCM, Triton injection resulted in a significant (p < 0.001) increase in plasma levels of total cholesterol,
triglycerides and LDL-C by about 33%, 80% and 54%, respectively (Fig. 3 (a, b)). In addition, Triton significantly (p < 0.001)
increased atherogenic index and LDL/HDL ratio. No significant (p > 0.05) difference in the level of HDL-C was observed
between NCM and hyperlipidemic groups (HCM and HCDM) (Fig. 3b).

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Fig. 3. Effect of the fractions on hyperlipidemia induced by Triton in mice. (a) Effect on plasma total cholesterol and triglycerides. (b) Effect on plasma
HDL-C and LDL-C. (c) Effect on atherogenic index and LDL-C/HDL-C ratio. AF: aqueous fraction treated mice, DCMF: dichloromethane fraction treated mice,
EAF: ethyl acetate fraction treated mice, Ff: fenofibrate treated mice, HCDM: hyperlipidemic + DMSO 4% control mice, HCM: hyperlipidemic control mice,
MF: methanol fraction treated mice, NCM: normolipidemic control mice. Results are expressed as the mean ± SD and analyzed with One-way ANOVA
followed by Tukey’s post hoc test, n = 10. ∗ p < 0.01, HCM and HCDM vs. NCM; DCMF vs. HDCM; EAF, MF, AF and Ff vs. HCM.

Table 4
Effect of the fractions derived from T. atlanticus on xylene-induced ear edema in mice.

Group Ear edema rate (%)

2h 4h 6h 8h 10 h

Negative control 28.89 ± 0.75 41.31 ± 0.79 41.30 ± 0.60 47.39 ± 0.50 37.32 ± 0.67
DCMF 21.61 ± 4 .00∗ 22.19 ± 0.85∗ 19.97 ± 2.56∗ 9.97 ± 2.56∗ 9.23 ± 0.62∗
EAF 24.63 ± 8.47 23.59 ± 3.22∗ 22.68 ± 3.47∗ 22.68 ± 3.47∗ 19.91 ± 4.74∗
MF 0∗ 24.60 ± 8.20∗ 30.95 ± 12.51 29.84 ± 9.84 23.61 ± 13.61∗
AF 28.33 ± 0.29 46.06 ± 10.54 40.70 ± 0.70 46.85 ± 2.47 46.85 ± 2.47
Acetylsalicylic acid 34.023 ± 0.95∗ 33.33 ± 1.36∗ 17.59 ± 0.72 15.51 ± 0.64∗ 11.57 ± 0.07∗

AF: aqueous fraction, DCMF: dichloromethane fraction, EAF: ethyl acetate fraction, MF: methanol fraction. The ani-
mals were treated with the fractions from T. atlanticus at 800 μg/ear. Acetylsalicylic acid was used as a positive con-
trol (400 μg/ear). Results are expressed as the mean ± SD and analyzed with one-way ANOVA followed by Tukey’s
post hoc test, (n = 6), ∗ p < 0.05 (vs. Negative control).

The administration of the fractions and fenofibrate prevented the changes of plasma lipid levels induced by Triton. Com-
pared with corresponding hyperlipidemic group, the fractions significantly (p < 0.001) decreased the plasma levels of total
cholesterol, triglycerides and LDL-C (Fig. 3 (a, b)), and produced a marked decrease of the AI and LDL-C/HDL-C ratio values
(Fig. 3c). Interestingly, AF effectively increased HDL-C level by about 43.87% compared to HCM (p < 0.01) (Fig. 3b).

Effect of the fractions on inflammation induced by xylene and AA

The anti-inflammatory activity was evaluated using xylene- and AA-induced edema models. As shown in Table 4, local
application of xylene produced a significant increase in the thickness of the mice ear (control).
Compared to control, simultaneous application of DCMF or EAF (800 μg/ear) with xylene inhibited the increase of
the edema thickness by about 79% and 52%, respectively, at 8 h after treatment. Acetylsalicylic acid applied at a dose of
400 μg/ear inhibited edema by about 67%.
The results of the anti-inflammatory effect of oral administration of the fractions on rat ear edema induced by xylene are
summarized in Table 5. The sup-plantar injection of AA induced edema within 30 min, peaking in intensity after 150 min
of injection. The oral administration of EAF and MF (25 mg/kg BW) effectively inhibited AA-induced edema (Table 5). After
150 min of treatment, EAF and MF inhibited edema formation by about 84% and 91%, respectively. MF and EAF produced
an anti-inflammatory effect higher than indomethacin, which reduced edema by about of 61% at 150 min, compared with
control. AF and DCMF did not show any effect.

8
T. Khouya, M. Ramchoun, A. Hmidani et al. Scientific African 11 (2021) e00716

Table 5
Effect of the fractions derived from T. atlanticus on arachidonic acid-induced paw edema in rats.

Group Paw edema inhibition % (Mean value ± SD)

30 min 60 min 90 min 120 min 150 min 180 min 210 min 240 min

DCMF 6.74 ± 0.60 6.24 ± 2.03 2.65 ± 1.45 5.07 ± 1.31 10.79 ± 2.03 1.05 ± 1.58 0 ± 1.11 15.01 ± 1.04
AEF 32.86 ± 0.45 61.33 ± 2.27 70.84 ± 0.91 76.98 ± 0.51 84.26 ± 0.33 87.28 ± 1.03 87.97 ± 0.62 86.15 ± 1.00
MF 65.38 ± 0.71 77.45 ± 1.20 78.12 ± 1.56 81.86 ± 1.98 91.02 ± 1.40 89.71 ± 1.40 95.36 ± 0.50 96.98 ± 0.69
FA 0 ± 0.82 0 ± 1.91 0 ± 1.12 0.54 ± 1.37 5.66 ± 1.64 0 ± 2.71 0 ± 2.12 8.79 ± 2.03
Indomethacin 0 ± 2.17 31.54 ± 1.84 54.00 ± 2.49 56.58 ± 3.25 60.92 ± 5.92 56.85 ± 5.46 60.32 ± 5.32 64.34 ± 4.48

AF: aqueous fraction, DCMF: dichloromethane fraction, EAF: ethyl acetate fraction, MF: methanol fraction. Rats were treated with the fractions from T.
atlanticus at 25 mg/kg. Indomethacin (5 mg/kg) was used as a positive control. Results are expressed as the mean ± SD, n = 8. Results are reported as
percentage of edema inhibition in treated groups as compared with control group.

Effect of the fraction on coagulation in vitro

The results of the effect of the fractions on coagulation in vitro are given in Tables 6, 7 and 8. The pre-incubation of EAF,
MF and FA with plasma induced a significant (p ˂ 0.001) prolongation of clotting time even at low concentrations in PT, PTT
and TT tests. In TT test, all three fractions at a concentration of 560 μg/mL completely inhibited clotting in blood plasma.
EAF and MF at 280 μg/mL resulted in complete inhibition of clotting in both PT and PTT tests. All fractions at 280 μg/mL
exhibited an anticoagulant effect higher than heparin (1 μg/mL).

Discussion

The development of CVDs implicates inflammation and coagulation systems as well as lipid metabolism abnormalities.
In the present study, the findings showed that the polyphenol-rich fractions of T. atlanticus exhibited significant antioxidant,
anti-inflammatory, anticoagulant and anti-hyperlipidemic activities.
Various phenolic compounds were detected in T. atlanticus fractions including caffeic acid, rosmarinic acid, quercetin,
rutin, hyperoside and luteolin-7-O-glucoside. Caffeic and rosmarinic acids were the more abundant phenolic compounds in
EAF and polar fractions (MF and AF). Rosmarinic acid has been reported to be the most abundant caffeic dimer in many
species of Thymus genus such as T. satureioides and T. zygis [21,24]. The differences observed in biological activities of T.
atlanticus fractions may be attributed to the variation in their phenolic composition [21].
Xylene and arachidonic acid-induced edema models are frequently used to explore the anti-inflammatory effect of the
compounds and extracts derived from plants [23,25]. The results obtained from these models demonstrated a potent pro-
tective effect of T. atlanticus against inflammation. EAF had the highest anti-inflammatory activity in both models. The anti-
inflammatory activity of the fractions correlates with the polyphenol content as the EAF and MF which had the highest
phenolic content showed the highest inhibitory effect on inflammation. However, despite its low polyphenol content, DCMF
was effective in reducing ear edema induced by xylene upon local treatment. This may be explained by the lipophilicity of
DCMF making it able to penetrate through the epidermal membrane following the external application. In accordance with
these findings, a previous study found that oral administration of EAE and EM effectively prevented inflammation provoked
by carrageenan and DCMF was effective in inhibiting inflammation mediated by croton oil in mice after local treatment
[16]. The carrageenan model is widely used to identify drugs that are inhibitors of the cyclooxygenase (COX) but it is also
sensitive to the dual 5-lypoxygenase (LOX)/COX inhibitors. The rat model of arachidonic acid-induced edema is sensitive to
5-LOX inhibitors. Thus, the anti-inflammatory action of EAF and MF could be attributed to a dual inhibition of LOX and COX.
Xylene-induced edema is associated with the release of substance P. Substance P is a member of tachykinin neuropep-
tides family, that can activate signaling events leading to neurogenic inflammation and swelling. Xylene also induces release
of pro-inflammatory mediators such as fibrinolysin, histamine and kinins. These two events are interconnected since sub-
stance P provokes the secretion of histamine which, in turn, induces the release of substance P [25]. The fact that croton
oil-induced ear edema is also associated with the release of histamine, the anti-inflammatory effect of T. atlanticus extracts
observed here and in previous studies [16,18] may be partially related to the inhibition of histamine. Many researchers have
confirmed that polyphenols are effective in inhibiting inflammation by attenuating the release of potent inflammatory me-
diators such as cytokines and chemokines, and by inhibiting key enzymes of inflammation (COX, 5-LOX) [26]. Inhibition of
these mediators plays an important role in the resolution of inflammation and the treatment of chronic inflammatory dis-
eases such as heart disease [27]. It has been reported that rosmarinic acid has potent anti-inflammatory effect and inhibits
the secretion of inflammatory mediators by inflammatory cell cultures and in animal models [18,28].
Rocha et al. [29] indicated that rosmarinic acid effectively inhibited carrageenan-induced acute inflammation in rat
model. Rosmarinic acid was the predominant phenolic acid in the active fractions of T. atlanticus. It is likely that the anti-
inflammatory activities of these fractions are related to the presence of rosmarinic acid. However, other phenolic compounds
found in these fractions have been reported to possess potent anti-inflammatory activity, including caffeic acid, quercetin,
rutin, hyperoside and luteolin-7-O-glucoside.

9
 T. Khouya, M. Ramchoun, A. Hmidani et al.
Table 6
Effect of the fractions of T. atlanticus on prothrombin time.

Fractions Prothrombin Time (s)

Control time (s) Heparin (1 μg/mL) Concentrations of fractions (μg/mL plasma)

17.5 35 70 140 280 560

10

MF 15.01 ± 0.24 60.03 ± 1.54∗ 14.17 ± 0.12 13.63 ± 0.34 15.93 ± 0.50 27.27 ± 0.58∗ 99.13 ± 1.34∗ ˃ 300∗
EAF 15.01 ± 0.25 60.03 ± 1.54∗ 26.62 ± 1.13∗ 42.32 ± 1.12∗ 110.79 ± 0.66∗ 170.36 ± 1.05∗ 310.62 ± 0.68∗ ˃ 300∗
AF 15.01 ± 0.26 60.03 ± 1.54∗ 23.16 ± 1.02∗ 24.11 ± 1.33∗ 25.14 ± 1.38∗ 30.45 ± 0.09∗ 47.25 ± 0.21∗ 62.11 ± 1.19∗

AF: aqueous fraction, MF: methanol fraction, EAF: ethyl acetate fraction. Concentrations are expressed as mg of dry extract per mL of clotting mixture. Heparin was
used as a positive control. Results are presented as the mean ± SD and analyzed with one-way ANOVA followed by Tukey’s post hoc test, n = 6. ∗ p < 0.001 vs. control.

Scientific African 11 (2021) e00716

 T. Khouya, M. Ramchoun, A. Hmidani et al.
Table 7
Effect of the fractions of T. atlanticus on thrombin time.

Fractions Thrombin Time (s)

Control time (s) Heparin (1 μg/mL) Concentrations of fractions (μg/mL plasma)

8.75 17.5 35 70 140 280 560

11

MF 17.44 ± 0.02 51.68 ± 0.83∗ 26.07 ± 0.06∗ 38.30 ± 0.23∗ 46.02 ± 0.02∗ 86.69 ± 0.33∗ 119.38 ± 0.35∗ > 300∗ > 300∗
EAF 17.44 ± 0.02 51.68 ± 0.83∗ 31.26 ± 0.18∗ 38.70 ± 0.03∗ 59.81 ± 0.63∗ 85.48 ± 0.30∗ 115.62 ± 0.06∗ 155.41 ± 0.06∗ > 300∗
AF 17.44 ± 0.02 51.68 ± 0.83∗ 31.98 ± 0.57∗ 57.59 ± 0.92∗ 74.83 ± 1.11∗ 103.13 ± 0.90∗ 116.20 ± 1.70∗ 246.65 ± 2.49∗ > 300∗

AF: aqueous fraction, EAF: ethyl acetate fraction, MF: methanol fraction. Concentrations are expressed as mg of dry extract per mL of clotting mixture. Heparin was used as a
positive control. Results are presented as the mean ± SD and analyzed with one-way ANOVA followed by Tukey’s post hoc test, n = 6. ∗ p < 0.001 vs. control.

Scientific African 11 (2021) e00716

 T. Khouya, M. Ramchoun, A. Hmidani et al.
Table 8
Effect of the fractions of T. atlanticus on partial thromboplastin time.

Fractions Partial Thromboplastin Time (s)

Control time (s) Heparin (1 μg/mL) Concentrations of fractions (μg/mL plasma)

8.75 17.5 35 70 140 280 560

12

MF 16.10 ± 0.15 310.10 ± 5.26∗ 18.07 ± 0.40∗ 20.97 ± 0.25∗ 29.47 ± 1.19∗ 60.87 ± 3.50∗ 300.10 ± 5.72∗ ˃ 900∗ ˃ 900∗
EAF 16.10 ± 0.15 310.10 ± 5.26∗ 18.50 ± 0.36∗ 24.44 ± 0.11∗ 40.14 ± 0.88∗ 100.74 ± 1.61∗ 160.31 ± 0.65∗ 300.57 ± 0.57∗ ˃ 900∗
AF 16.10 ± 0.15 310.10 ± 5.26∗ 19.86 ± 1.19 19.86 ± 2.01 20.81 ± 1.33∗ 21.84 ± 1.19∗ 27.15 ± 1.09∗ 43.95 ± 1.19∗ 58.81 ± 1.02∗

AF: aqueous fraction, EAF: ethyl acetate fraction, MF: methanol fraction. Concentrations are expressed as mg of dry extract per mL of clotting mixture. Heparin was used as a positive
control. Results are presented as the mean ± SD and analyzed with one-way ANOVA followed by Tukey’s post hoc test, n = 6. ∗ p < 0.001 vs. control.

Scientific African 11 (2021) e00716

T. Khouya, M. Ramchoun, A. Hmidani et al. Scientific African 11 (2021) e00716

Polyphenols are well known to have antioxidant activities [30]. Oxidative stress play an important role in the de-
velopment of inflammation and it is related to various diseases [31]. In the present study, the fractions of T. atlanticus
showed potent antioxidant activity comparing to standard antioxidant (BHT), as evidenced by ABTS radical scavenging, β -
carotene/linoleic acid and MDA assays. The polyphenol contents of the fractions were positively correlated with the antiox-
idant activity in ABTS (r = 0.8) and β -carotene/linoleic (r = 0.9) assays. The anti-inflammatory properties of polyphenols
are partly due to their ability to scavenge reactive oxygen species (ROS) and to inhibit ROS release from the inflammatory
cells [32]. This may partially explain the efficacy of the active fractions of T. atlanticus in alleviating inflammation induced
by xylene and arachidonic acid in animal models.
Inflammation is closely linked with blood coagulation in pathological conditions [33]. Moreover, a large number of in-
flammatory mediators are known to activate platelets and stimulate the release of coagulation factors. The latter factors,
in turn, evoke mediators release from the pro-inflammatory cells [34]. Most clinical studies have confirmed the beneficial
role of the use of anticoagulants in several chronic diseases including ischemic heart disease and stroke [35]. However, the
benefits of oral anticoagulants are controversial. Their use is associated with major bleeding events [11]. Plants may provide
a source of natural anticoagulants with fewer side effects and that are effective in preventing thrombosis. The rabbit is the
most used animal model for screening anticoagulant compounds and the results of such studies are effective to conclude the
plasma levels of anticoagulant required for therapeutic activity in humans. In the present study, blood of rabbits was used to
evaluate the in vitro anticoagulant effect of the fractions by using three tests: TP, PTT and TT, which are often used in clin-
ical practice to detect coagulation abnormalities and screen new anticoagulants [18]. All tested fractions (MF, EAF and AF)
affected coagulation through inhibition of extrinsic, intrinsic and common pathways. A previous study using rat blood indi-
cated that aqueous extract of T. atlanticus inhibited both extrinsic and intrinsic pathways of coagulation [16]. It was recently
noticed that polyphenol-rich fractions of T. satureioides of the Moroccan High Atlas showed a marked pro-coagulant effect
on rat blood [21]. The differences observed between Thymus varieties may probably due to variation in phenolic composition
[16]. However, the mechanism of the anticoagulant action is not explored at this stage as well as the findings concerning
the anticoagulant effect of different phenolic acids identified in T. atlanticus fractions are less available in literature.
In addition, we evaluated the effect of the fractions on hyperlipidemia induced by Triton in mice. The injection of Tri-
ton induced lipid profile changes similar to those described in literature [36]. This evidenced by significant increase in
plasma levels of total cholesterol, TG and LDL-C, which are considered as major risk factors of atherosclerosis and CVDs
[37]. Moreover, modified LDL-C particles induce many atherogenic responses including secretion of inflammatory mediators
and endothelial dysfunction [37]. Pharmaceutical reduction of serum lipid concentrations is the main strategy for the control
of CVDs [38]. Despite their efficacy, current anti-hyperlipidemic drugs have many adverse effects [39]. On the other hand,
habitual intake of polyphenol-rich foods has been associated with reduced incidence of CVDs [4]. In the present study, the
administration of a single dose of the fractions at 2 g/kg BW decreased total cholesterol, TG and LDL-C in mice injected
with Triton. Similar results were found in the group receiving fenofibrate at 65 mg/kg BW. In addition, the aqueous fraction
of T. atlanticus effectively increased HDL-C level in hyperlipidemic mice. This suggested an additional beneficial effect of T.
atlanticus fractions since low plasma HDL-C levels are associated with the risk of various diseases such as coronary artery
disease and autoimmune diseases [40]. It is clear that all fractions of T. atlanticus and fenofibrate exert similar effect on
hyperlipidemia induced by Triton in mice. The dose of the fractions and fenofibrate was chosen based on the findings of a
previous study [17]. The latter study [17] demonstrated that aqueous extract of T. atlanticus ameliorated acute hyperlipidemia
in rats. However, compared to rat, the mouse is the useful animal model for studying hyperlipidemia since it has many sim-
ilarities with the human concerning the lipid metabolism. Thus, the present findings provide clear evidence regarding the
anti-hyperlipidemic effect of T. atlanticus. Although there is no significant correlation between anti-hyperlipidemic effect
and polyphenol content of the fractions, polyphenols may be responsible for the anti-hyperlipidemic activities of T. atlanti-
cus as they have been reported to modulate hyperlipidemia through inhibition of cholesterol synthesis and improvement of
cholesterol efflux and bile acid synthesis [26].
In view of the above discussion, T. atlanticus may be a rich source of biologically active polyphenols as this herb ap-
pears to have antioxidant, anticoagulant, anti-hyperlipidemic and anti-inflammatory activity. However, additional studies are
needed to isolate the active compounds and to identify the exact mechanisms of actions.
The present study validates the use of T. atlanticus in traditional medicine as remedy of chronic inflammatory diseases.
Phytochemical analysis showed that T. atlanticus is rich in phenolic compounds that might be responsible for its pharmaco-
logical properties.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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T. Khouya, M. Ramchoun, A. Hmidani et al. Scientific African 11 (2021) e00716

Author contribution

TK, MR, CA and SA conceived the idea. TK, EDTB and AH developed the manuscript and discussed findings. TK, RM, EDTB
and AH carried out the experiments. TK wrote the manuscript. CA and MR provided revisions to the manuscript. Final copy
was read and approved by all authors.

Acknowledgment

The authors would like to thank Dr. Mohamed IBN TATTOU (Scientific Institute at the University of Mohammed V. Ra-
bat, 10170, Morocco) for plant identification. The authors also thank Pr. Hicham HARNAFI, Pr. Mohamed BENLYAS and Dr.
Benaïssa EL MOUALIJ for their valuable guidance that helped improve the quality of the research.

References

[1] J. Bellakhdar, R. Claisse, J. Fleurentin, C. Younos, Repertory of standard herbal drugs in the Moroccan pharmacopoea, J. Ethnopharmacol. 35 (1991)
123–143, doi:10.1016/0378-8741(91)90064-K.
[2] B. Salehi, M.S. Abu-Darwish, A.H. Tarawneh, C. Cabral, A.V. Gadetskaya, L. Salgueiro, T. Hosseinabadi, S. Rajabi, W. Chanda, M. Sharifi-Rad, R.B. Mulaudzi,
S.A. Ayatollahi, F. Kobarfard, D.K. Arserim-Uçar, J. Sharifi-Rad, A. Ata, N. Baghalpour, M.del M. Contreras, Thymus spp. plants - food applications and
phytopharmacy properties, Trends Food Sci. Technol. 85 (2019) 287–306, doi:10.1016/j.tifs.2019.01.020.
[3] J. Wang, J. Xu, X. Gong, M. Yang, C. Zhang, M. Li, J. Wang, J. Xu, X. Gong, M. Yang, C. Zhang, M. Li, Biosynthesis, chemistry, and pharmacology of
polyphenols from chinese salvia species: a review, Molecules 24 (2019) 155, doi:10.3390/molecules24010155.
[4] R.D. Mendonça, N.C. Carvalho, J.M. Martin-Moreno, A.M. Pimenta, A.C.S. Lopes, A. Gea, M.A. Martinez-Gonzalez, M. Bes-Rastrollo, Total polyphenol
intake, polyphenol subtypes and incidence of cardiovascular disease: The SUN cohort study, Nutr. Metab. Cardiovasc. Dis. 29 (2019) 69–78, doi:10.
1016/j.numecd.2018.09.012.
[5] Cardiovascular diseases (CVDs), (n.d.). https://www.who.int/news-room/fact-sheets/detail/cardiovascular- diseases- (cvds) (Accessed April 16, 2019).
[6] J.R. Burnett, A.J. Hooper, R.A. Hegele, Lipids and cardiovascular disease, Pathology 51 (2019) 129–130, doi:10.1016/j.pathol.2018.12.001.
[7] L. Ferrucci, E. Fabbri, Inflammageing: chronic inflammation in ageing, cardiovascular disease, and frailty, Nat. Rev. Cardiol. 15 (2018) 505–522, doi:10.
1038/s41569- 018- 0064- 2.
[8] A. Tournadre, Statins, myalgia, and rhabdomyolysis, Jt. Bone Spine (2019) 1–6, doi:10.1016/j.jbspin.2019.01.018.
[9] V. Neergheen, A. Dyson, L. Wainwright, I.P. Hargreaves, Statin and fibrate-induced dichotomy of mitochondrial function, in: Mitochondrial Dysfunction
Caused by Drugs and Environmental Toxicants, Wiley, 2018, pp. 457–473, doi:10.1002/9781119329725.ch30.
[10] S.J. Kottoor, R.R. Arora, The utility of anti-inflammatory agents in cardiovascular disease: a novel perspective on the treatment of atherosclerosis, J.
Cardiovasc. Pharmacol. Ther. 23 (2018) 483–493, doi:10.1177/1074248418778548.
[11] L.A. Ferri, G. Bassanelli, S. Savonitto, Use of direct oral anticoagulant in ischaemic heart disease: the COMPASS study, Eur. Hear. J. Suppl. 21 (2019)
B84–B87, doi:10.1093/eurheartj/suz003.
[12] J. Sakaki, M. Melough, S.G. Lee, G. Pounis, O.K. Chun, Polyphenol-Rich Diets in Cardiovascular Disease Prevention, Elsevier Inc., 2018, doi:10.1016/
b978- 0- 12- 814556- 2.0 0 010-5.
[13] P. Rohdewald, Pleiotropic effects of french maritime pine bark extract to promote healthy aging, Rejuvenation Res. 22 (2019) 210–217, doi:10.1089/rej.
2018.2095.
[14] A. Benabid, Flore et écosystèmes du Maroc : évaluation et préservation de la biodiversité, Ibis Press, 20 0 0.
[15] R. Morales, The History, Botany and Taxonomy of the Genus Thymus. In: Stahl-Biskup E., Sáez F., Eds., Thyme: The Genus Thymus, 1st, Taylor and
Francis, Inc., London, 2002, pp. 1–43.
[16] T. Khouya, M. Ramchoun, A. Hmidani, S. Amrani, H. Harnafi, M. Benlyas, Y.F. Zegzouti, C. Alem, Anti-inflammatory, anticoagulant and antioxidant effects
of aqueous extracts from Moroccan thyme varieties, Asian Pac. J. Trop. Biomed. 5 (2015), doi:10.1016/j.apjtb.2015.05.011.
[17] M. Ramchoun, H. Harnafi, C. Alem, B. Büchele, T. Simmet, M. Rouis, F. Atmani, S. Amrani, Hypolipidemic and antioxidant effect of polyphenol-rich
extracts from Moroccan thyme varieties, ESPEN. J. 7 (2012) 3–8, doi:10.1016/j.clnme.2012.02.005.
[18] T. Khouya, M. Ramchoun, S. Amrani, H. Harnafi, M. Rouis, D. Couchie, T. Simmet, C. Alem, Anti-inflammatory and anticoagulant effects of polyphenol-
rich extracts from Thymus atlanticus: An in vitro and in vivo study, J. Ethnopharmacol. 252 (2020), doi:10.1016/j.jep.2019.112475.
[19] D. Heimler, P. Vignolini, M.G. Dini, F.F. Vincieri, A. Romani, Antiradical activity and polyphenol composition of local Brassicaceae edible varieties, Food
Chem. 99 (2006) 464–469, doi:10.1016/J.FOODCHEM.2005.07.057.
[20] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Antioxidant activity applying an improved ABTS radical cation decolorization
assay, Free Radic, Biol. Med. 26 (1999) 1231–1237, doi:10.1016/S0891-5849(98)00315-3.
[21] T. Khouya, M. Ramchoun, A. Hmidani, B. El moualij, S. Amrani, H. Harnafi, M. Benlyas, Y.F. Zegzouti, E.H. Nazih, K. Ouguerram, C. Alem, Acute toxicity
and antiproliferative and procoagulant activities of fractions derived from Thymus satureioides of the Moroccan High Atlas, South African J. Bot. 121
(2019) 568–576, doi:10.1016/j.sajb.2019.01.005.
[22] X.C. Tang, Z.G. Lin, W. Cai, N. Chen, L. Shen, Anti-inflammatory effect of 3-acetylaconitine, Zhongguo Yao Li Xue Bao 5 (1984) 85–89 http://www.ncbi.
nlm.nih.gov/pubmed/6235717. (accessed April 17, 2019).
[23] M.J. DiMartino, G.K. Campbell, C.E. Wolff, N. Hanna, The pharmacology of arachidonic acid-induced rat paw edema, Agents Actions 21 (1987) 303–305
http://www.ncbi.nlm.nih.gov/pubmed/3120509. (accessed April 17, 2019).
[24] T. Khouya, M. Ramchoun, A. Hmidani, S. Amrani, H. Harnafi, M. Benlyas, Y. Filali Zegzouti, C. Alem, Chemical characterization and evaluation of
antioxidant, anti-inflammatory and anticoagulant activity of aqueous extract and organic fractions of thymus Zygis L. sub SP. Gracilis, Int. J. Pharm. Sci.
Res. 7 (2016) 1396–1405, doi:10.13040/IJPSR.0975- 8232.7(4).1396- 05.
[25] F. Peng, Y. Xie, X. Li, G. Li, Y. Yang, Chemical components and bioactivity evaluation of extracts from pear (Pyrus Ussuriensis Maxim) fruit, J. Food
Biochem. 42 (2018) 1–9, doi:10.1111/jfbc.12586.
[26] M.-L. Ricketts, B.S. Ferguson, Polyphenols: Novel signaling pathways, Curr. Pharm. Des. 24 (2018) 158–170, doi:10.2174/1381612824666171129204054.
[27] J.N. Fullerton, D.W. Gilroy, Resolution of inflammation: a new therapeutic frontier, Nat. Rev. Drug Discov. 15 (2016) 551–567, doi:10.1038/nrd.2016.39.
[28] C. Colica, L. Di Renzo, V. Aiello, A. De Lorenzo, L. Abenavoli, Rosmarinic acid as potential anti-inflammatory agent, Rev. Recent Clin. Trials 13 (2018)
240–242, doi:10.2174/157488711304180911095818.
[29] J. Rocha, M. Eduardo-Figueira, A. Barateiro, A. Fernandes, D. Brites, R. Bronze, C.M. Duarte, A.T. Serra, R. Pinto, M. Freitas, E. Fernandes, B. Silva-Lima,
H. Mota-Filipe, B. Sepodes, Anti-inflammatory effect of Rosmarinic acid and an extract of Rosmarinus officinalis in Rat models of local and systemic
inflammation, Basic Clin. Pharmacol. Toxicol. 116 (2015) 398–413, doi:10.1111/bcpt.12335.
[30] M. Bijak, J. Saluk, R. Szelenberger, P. Nowak, Popular naturally occurring antioxidants as potential anticoagulant drugs, Chem. Biol. Interact. 257 (2016)
35–45, doi:10.1016/j.cbi.2016.07.022.
[31] U. Förstermann, N. Xia, H. Li, Roles of vascular oxidative stress and nitric oxide in the pathogenesis of atherosclerosis, Circ. Res. 120 (2017) 713–735,
doi:10.1161/CIRCRESAHA.116.309326.

14
T. Khouya, M. Ramchoun, A. Hmidani et al. Scientific African 11 (2021) e00716

[32] H. Asakura, T. Kitahora, Antioxidants and polyphenols in inflammatory bowel disease: ulcerative colitis and Crohn disease, in: Polyphenols: Prevention
and Treatment of Human Disease, Elsevier, 2018, pp. 279–292, doi:10.1016/B978- 0- 12- 813008- 7.00023- 0.
[33] Y. Wu, Contact pathway of coagulation and inflammation, Thromb. J. (2015), doi:10.1186/s12959- 015- 0048- y.
[34] C.B. Keragala, D.F. Draxler, Z.K. McQuilten, R.L. Medcalf, Haemostasis and innate immunity – a complementary relationship: A review of the intricate
relationship between coagulation and complement pathways, Br. J. Haematol. 180 (2018) 782–798, doi:10.1111/bjh.15062.
[35] T.Y. Chang, J.N. Liao, T.F. Chao, J.J. Vicera, C.Y. Lin, T.C. Tuan, Y.J. Lin, S.L. Chang, L.W. Lo, Y.F. Hu, F.P. Chung, S.A. Chen, Oral anticoagulant use for stroke
prevention in atrial fibrillation patients with difficult scenarios, IJC Hear. Vasc. 20 (2018) 56–62, doi:10.1016/j.ijcha.2018.08.003.
[36] E.dine T. Bouhlali, A. Hmidani, B. Bourkhis, T. Khouya, H. Harnafi, Y. Filali-Zegzouti, C. Alem, Effect of Phoenix dactylifera seeds (dates) extract in triton
WR-1339 and high fat diet induced hyperlipidaemia in rats: a comparison with simvastatin, J. Ethnopharmacol. 259 (2020) 112961, doi:10.1016/j.jep.
2020.112961.
[37] A.J. Kattoor, S.H. Kanuri, J.L. Mehta, Role of Ox-LDL and LOX-1 in atherogenesis, Curr. Med. Chem. 25 (2018), doi:10.2174/0929867325666180508100950.
[38] S.O. Almeida, M. Budoff, Effect of statins on atherosclerotic plaque, Trends Cardiovasc. Med. (2019), doi:10.1016/j.tcm.2019.01.001.
[39] S. Sultan, A. D’Souza, I. Zabetakis, R. Lordan, A. Tsoupras, E.P. Kavanagh, N. Hynes, Statins: rationale, mode of action, and side effects, in: Impact of
Nutrition and Statins on Cardiovascular Diseases, Elsevier, 2019, pp. 171–200, doi:10.1016/B978- 0- 12- 813792- 5.0 0 0 06-9.
[40] C.M. Madsen, A. Varbo, B.G. Nordestgaard, Low HDL Cholesterol and high risk of autoimmune disease: two population-based cohort studies including
117341 individuals, Clin. Chem. 65 (2019) 644–652, doi:10.1373/clinchem.2018.299636.

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