Received: 14 October 2021 | Revised: 7 March 2022 | Accepted: 4 April 2022

DOI: 10.1111/jfbc.14225

ORIGINAL ARTICLE

Effect of supplementation with polyphenol extract of Thymus

atlanticus on paraoxonase-­1 activity, insulin resistance, and
lipid profile in high-­fat diet-­fed hamsters

Tarik Khouya1 | Mhamed Ramchoun1,2,3 | Abdelbassat Hmidani1 | Souliman Amrani3 |

Mohamed Benlyas4 | Fatima Kasbi Chadli5 | Khadija Ouguerram6 | Chakib Alem1

1
Team of Biochemistry and Natural
Substances, Faculty of Sciences and Abstract
Techniques, Moulay Ismail University,
Thymus atlanticus has been used by Moroccan people to treat a variety of health prob-
Errachidia, Morocco
2
Laboratory of Biotechnology and
lems, particularly metabolic disorders. In this study, hamsters fed a high-­fat diet daily
Sustainable Development of Natural received distilled water (a positive control) or a single dose of Thymus atlanticus poly-
Resources, Polydisciplinary Faculty,
University Sultan Moulay Slimane, Beni
phenols (Pp) for 63 days. The negative control was fed a normal diet and received
Mellal, Morocco distilled water. Results showed that the supplementation of HFD with Pp significantly
3
Laboratory of Biochemistry and (p < .001) reduced the levels of MDA and LDL cholesterol, restored insulin level, and
Biotechnologies, Faculty of Sciences,
University Mohamed I, Oujda, Morocco increased the activities of serum paraoxonase-­1 and HDL cholesterol levels, but did
4
Department of Biology, Faculty of not affect (p > .05) the activity of superoxide dismutase and glutathione peroxidase
Science, Moulay Ismail University,
when compared with the group feeding HFD alone. Thymus atlanticus could be an ef-
Meknes, Morocco
5
INRAe UMR1280 PhAN, Physiopathology fective agent against dyslipidemia, oxidative stress, and insulin resistance.
of Nutritional Adaptations, CHU Hôtel
Dieu, IMAD, CRNH Ouest, University of
Practical applications
Nantes, Nantes Cedex, France HFD consumption is a risk factor for oxidative stress and the development of meta-
6
UMR1280 PhAN, Physiopathology bolic disorders, such as hyperlipidemia and insulin resistance, which may result in ath-
of Nutritional Adaptations, INRAe,
University of Nantes, CHU Hôtel Dieu, erosclerosis and related cardiovascular diseases, the leading causes of death globally.
IMAD, CRNH Ouest, Nantes, France The management of these alterations is an important strategy to prevent and treat
Correspondence heart complications. Our results showed thatT. atlanticus effectively alleviated HFD-­
Tarik Khouya, Department of Biology, induced hyperlipidemia and insulin resistance and improved PON1 activity. T. atlan-
Faculty of Sciences and Techniques,
University Moulay Ismail, Errachidia ticus is a source of biomolecules that may be an effective supplement for controlling
52000, Morocco. HFD-­related metabolic disorders. Therefore, the findings of this study may be helpful
Email: tarikkhouya@yahoo.com
in the preparation of effective supplements from T. atlanticus to control metabolic
Funding information disorders and related complications.
The authors declare that there was no
specific funding for the present research.
KEYWORDS
high-­fat diet, insulin resistance, oxidative stress, Paraoxonase-­1, Thymus atlanticus

1 | I NTRO D U C TI O N
The consumption of high-­fat diets is associated with oxidative stress
and metabolic disorders, such as hyperlipidemia and insulin resis-
tance (Duan et al., 2018). Hyperlipidemia causes oxidative stress as a
consequence of increased reactive oxygen species (ROS) production
during mitochondrial fatty acid β-­oxidation (Lian et al., 2020). Most
studies have reported that oxidative conditions are associated with
several diseases and, together with hyperlipidemia, constitute two
major factors involved in atherosclerosis and cardiovascular compli-
cations, such as stroke and coronary heart disease (Sies et al., 2017).
Increased plasma lipid levels harm glucose metabolism and lead to

J Food Biochem. 2022;00:e14225. wileyonlinelibrary.com/journal/jfbc © 2022 Wiley Periodicals LLC. | 1 of 11

https://doi.org/10.1111/jfbc.14225
2 of 11 | KHOUYA et al.
the development of insulin resistance, which is characterized pri-
marily by hyperinsulinemia combined with hyperglycemia (Duan
et al., 2018). Available literature has confirmed that oxidative stress
is heavily implicated in the induction of insulin resistance by affect-
ing insulin receptor signal transduction, resulting in decreased ex-
pression of cellular glucose transporters (Hurrle & Hsu, 2017).
Hyperlipidemia is characterized by an elevated amount of low-­
density lipoprotein (LDL) and a decreased concentration of high-­density
lipoprotein (HDL) in the blood. Under oxidative stress conditions, LDL
cholesterol (LDL-­C) particles can be modified to oxidized LDL-­C, which
are internalized by macrophages via receptors not inhibited by intra-
cellular cholesterol content. The accumulation of cholesterol induces
the transformation of macrophages into foam cells, which form the
main cellular component of atherosclerotic plaque (Wu et al., 2018).
Elevated levels of oxidized LDL-­C have been reported to correlate
with insulin resistance (Carantoni et al., 1998). On the contrary, HDL
particle concentrations are inversely associated with insulin resistance
and have been reported to have numerous potentially antiatherogenic
functions, including the improvement in cholesterol efflux, promotion
of endothelial and vascular function, inflammation inhibition, and anti-
oxidative properties (Lin et al., 2018; Nagao et al., 2018). Recent stud-
ies have demonstrated that the antiatherogenic and cardioprotective
properties of HDL result from the activity of its associated enzymes,
particularly paraoxonase-­1 (PON1) (Chistiakov et al., 2017). This en-
zyme is an esterase calcium-­
dependent glycoprotein belonging to
the PON family (PON1, PON2, and PON3). PON1 has arylesterase
activity (aromatic carboxylic acid ester hydrolysis), lactonase activity
(lactone hydrolysis), and paraoxonase activity (organophosphate hy-
drolysis) (Chistiakov et al., 2017). These activities support many other
physiological functions of PON1 other than its potent antioxidant
activity. This protein prevents the oxidation process of lipoproteins,
especially LDL-­C. Moreover, PON1 deficiency is correlated with in-
creased LDL-­C oxidation and accentuation of atherosclerosis (Kotur-­
Stevuljević et al., 2020).
A decrease in PON1 concentration or activity in the blood has
been found in high oxidative stress conditions and subjects with
metabolic diseases, such as diabetes or hyperlipidemia, and aged
people (Mackness & Mackness, 2015). In addition, low PON1 activ-
ity has been found to lead to an increase in cholesterol levels and is
associated with a pro-­inflammatory process related to cardiovascu-
lar risk (Marek et al., 2018).
Pharmaceutical intervention to modulate gene expression or
PON1 catalytic activity has been proposed as a promising strategy
for preventing, controlling, or treating heart diseases and other se-
vere diseases, like metabolic disorders, degenerative diseases, and
chronic liver and renal failure. (Costa et al., 2005).
Other enzymes that play important roles in antioxidant defense
include glutathione peroxidase (GPx) and superoxide dismutase
(SOD), and measuring their activity is a helpful way to assess antioxi-
dant levels and oxidant stress in humans (Lei et al., 2021). Antioxidant
supplements may be beneficial in many conditions where the natu-
ral antioxidant defense is impaired (Sies et al., 2017). Polyphenols
are phytochemicals that are well known as potent antioxidants,
activators of antioxidant enzymes, such as PON1, and show a wide
range of biological activities (Martini et al., 2017).
The thymus is one of the most interesting plant genera as a
source of polyphenols with several properties, including analgesic,
antioxidant, anticoagulant, anti-­
inflammatory, antihyperlipidemic,
and cytotoxic activity, among others (Hmidani et al., 2019; Khouya
et al., 2019, 2021). Thymus atlanticus is a native plant of Morocco.
This herb grows in the mountains of the High Moroccan Atlas at al-
titudes of up to 2000 m. The stems and leaves of T. atlanticus have
been used for many centuries as a traditional treatment for a wide
variety of diseases, such as digestive diseases, and as an antibacte-
rial agent (Khouya et al., 2021).
The results from our previous studies reported that the aqueous
extract of T. atlanticus is rich in polyphenols with rosmarinic acid as
the most abundant compound (Khouya et al., 2015). Studies using
different animal models have shown that the administration of the
polyphenol-­rich extract (Pp) of T. atlanticus ameliorated Triton-­induced
acute hyperlipidemia in mice, rats, and hamsters, and chronic hyper-
lipidemia in mice and hamsters (Ramchoun et al., 2012; Ramchoun,
Khouya, Harnafi, Alem, et al., 2020; Ramchoun, Khouya, Harnafi,
Amrani, et al., 2020). Moreover, Pp administration reduced plasma
cholesterol and LDL-­C and both plasma and liver TG in hyperlipidemic
animals and, in some cases, produced a slight but significant eleva-
tion in HDL cholesterol (HDL-­C) (Ramchoun et al., 2012, Ramchoun,
Khouya, Harnafi, Alem, et al., 2020, Ramchoun, Khouya, Harnafi,
Amrani, et al., 2020). A potential mechanism of the lipid-­lowering ef-
fect of Pp is the inhibition of cholesterol biosynthesis by downregu-
lating 3-­hydroxy-­3-­methyl-­glutaryl-­coenzyme A reductase expression
without affecting cholesterol exertion (Ramchoun, Khouya, Alibrahim,
Hmidani, et al., 2020). Crude aqueous and organic extracts have
shown potent antioxidant activities in many studies in vitro (Hmidani
et al., 2019; Khouya et al., 2015). Additionally, our previous studies
have reported that Pp inhibited inflammation provoked by local ap-
plication of croton oil or xylene, and injection of carrageenan or ara-
chidonic acid in animal models (Khouya et al., 2015, 2021). It was also
found that Pp dose dependently inhibited the production of monocyte
chemoattractant protein 1 (MCP-­1) by macrophage cultures activated
by lipopolysaccharides (Khouya et al., 2020). An in vivo antioxidant
effect of Pp in acute hyperlipidemia has been reported (Ramchoun,
Khouya, Harnafi, Amrani, et al., 2020). However, the effect on chronic
hyperlipidemia has not yet been elucidated.
The goal of this study is to understand how the supplementa-
tion of a high-­fat diet (HFD) with Pp affects the lipoprotein profile,
plasma antioxidants, and glycemia and insulin levels in hamsters.
2 | M E TH O D S
2.1 | Reagents and materials
The rat insulin ELISA kit was obtained from Shibayagi Co., Ltd.,
(Gunma, Japan). Paraoxon and phenylacetate were bought from
Sigma-­Aldrich (St. Louis, MO, USA). All the other reagents were
KHOUYA et al.
TA B L E 1 Composition of experimental
diets
Standard diet
HFD
bought from bioMerieux (Paris, France). The glucometer was pur-
chased from Roche Diagnostics (Paris, France). Lipoprotein separa-
tion was carried out using fast protein liquid chromatography (AKTA
FPLC SYSTEM, GE Healthcare, USA).
2.2 | Plant
T. atlanticus aerial parts were collected in April from the High Atlas
in Morocco's Errachidia Province (32° 15’ N, 5° 25′ E, 2004 m). The
samples from the plant have been authenticated by Dr Ibn Tatou. A
specimen of the plant (voucher number: RAB 77497) has been de-
posited in the herbarium institute at the University of Mohammed
V, Rabat, Morocco.
2.3 | Preparation of polyphenol-­rich extract
A Pp was prepared from the T. atlanticus aerial parts as previously
described (Ramchoun et al., 2012). Briefly, about 100 g of powdered
leaf material was extracted for 4–­6 h with a Soxhlet extractor. The
extract solution was filtered to eliminate the nonsoluble fraction,
and the solvent was evaporated under a vacuum at 60 °C using a
rotator evaporator.
2.4 | Total phenolic content (TPC) assay
The determination of TPC was assessed by the Folin–­Ciocalteu assay
(Khouya et al., 2015). The reaction mixture included 30 μl of the sam-
ple, 500 μl of 10% sodium carbonate, and 250 μl of Folin–­Ciocalteu,
and the volume was completed to 5 ml with distilled water. The mix-
ture was then mixed. After 30 min, optical density was monitored at
725 nm, and TPC was expressed as mg caffeic acid equivalent (CAE)
per g of dried extract.
2.5 | Animals
Golden Syrian hamsters, 8 weeks of age, weighing between 70 and
90 g, obtained from the Animal Experimental Station in Nantes,
France, were used in this study. Animal experimentation was con-
ducted according to animal welfare laws and guidelines. Animal pro-
tocols were approved by the University of the local Animal Use and
Care Advisory Committee (Bretagne-­Pays de la Loire committee)
and conform to Directive 2010/63/EU.
| 3 of 11
Lipid
Proteins Starch Cellulose mixture Minerals Vitamins
23% 58% 6% 5% 8.5% 1.2%
24.7% 38.7% 6% 21% 8.5% 1.2%
2.6 | Diets
Two different diets, a standard diet and a high-­fat diet (HFD), were
employed in this experiment. The composition is given in Table 1.
The lipid mixture consisted of 3% palm oil and 2% corn oil for the
standard diet, and 3.5% palm oil, 15% lard oil, and 2.5% corn for the
HFD. All hamsters received the standard diet for 1 week of acclima-
tion before being fed an appropriate diet for 63 days.
2.7 | Experimental design
For 63 days, three groups of hamsters (n = 10) were studied. The
control group was maintained on a standard diet and administered
1.5 ml of distilled water (DW) by oral route. The hyperlipidemic
group (HF)-­fed HFD daily received DW. The treated group (HF-­Pp)
was fed with HFD and received a single dose of Pp (40 mg/100 g
body weight [BW]) every day for 63 days. Pp was dissolved in dis-
tilled water and given orally every morning at 8 a.m., for 63 days.
After the treatment, hamsters were fasted for 18 h and the blood
samples were collected in heparinized tubes after retro-­orbital
puncture under slight isoflurane anesthesia to determine glycemic
parameters, lipoprotein profile, and the activity of serum antioxi-
dant enzymes. The used dose of plant extract and the period of
study were chosen based on previous studies (Ramchoun, Khouya,
Alibrahim, Hmidani, et al., 2020; Ramchoun, Khouya, Harnafi,
Amrani, et al., 2020).
2.8 | Biochemical parameter examination
2.8.1 | Glycemia and insulin
The amount of fasting glucose was determined using a glucometer
(Roche Diagnostics, France). Plasma insulin concentration was ana-
lyzed using a rat insulin ELISA kit.
2.8.2 | Lipoprotein separation
Fast protein liquid chromatography (FPLC) was used according to the
method of Chétiveaux et al. (2002) to obtain the chromatograms of
plasma lipoproteins. The apparatus is equipped with a plus controller
(LCC-­501), a fraction collector (FRAC-­100), an ultraviolet monitor, a
multi-­injector (MV-­7, 200 μl), and double pumps (P-­500, 10 bar). The
absorbance was recorded at 280 nm.
4 of 11 | KHOUYA et al.
Elution was carried out in NaN3 (0.02%, pH 8.2) buffer, EDTA
(1 mM), and NaCl (154 mM). To filter the buffer solution, a 0.22-­μ m
filter (Duropore® GV filter, Millipore, Bedford, MA) was used. The
column was cleaned with water: ethanol (v: v, 80/20) solution. A
volume of 200 μl plasma was injected. The flow was set at 0.35 ml/
min for the elution. A complete profile was done after 105 min of
elution.
2.9 | Antioxidant activity in vivo
2.9.1 | Dosage of MDA
The amounts of MDA in the hamster plasma were determined accord-
ing to the method of Draper and Hadley (1990). The test tubes, con-
taining 150 μl of diluted serum, 0.35 ml of thiobarbituric acid (0.6%,
w/v), and 0.80 ml of phosphoric acid 1%, were placed in a water bath
for about half-­hour. After a rapid cooling on ice, 2 ml of n-­butanol was
added. The tubes were then centrifugated for 10 min at 3000 g, and
the optical density of the supernatant was monitored at 532 nm.
2.9.2 | Dosage of SOD
The SOD activity was measured following the previously reported
method with minor modifications (Sun et al., 1988). The mix-
ture reaction contained 40 μl of serum, 100 mM of PBS (pH 7.8),
240 mM of L-­m ethionine, 20 ml of EDTA, 1.4 mM of nitroblue tetra-
zolium, and 20 μM of riboflavin, and was incubated for 5 min under
4000 lx. The same mixture was prepared but placed away from
the light to serve as a blank. The optical density was recorded at
560 nm. The activity of SOD was calculated (Bouabid et al., 2020),
and one unit of SOD activity is expressed as the amount of en-
zyme able to inhibit the reduction of nitroblue tetrazolium by 50%
reduction (Sun et al., 1988).
2.9.3 | GPx activity
The determination of the GPx activity in hamster plasma was per-
formed according to the slightly modified method of Xu et al., 2017.
The enzyme was prepared by dissolving 2.4 U in 1 ml of potassium
phosphate buffer (0.15 mM). The activity of 2 U/ml GPx was deter-
mined in a reaction mixture composed of the tampon (pH 7), enzyme
cofactor (1 mM glutathione), and 0.15 mM of triphosphopyridine nu-
cleotide. Tampon was made by mixing 0.4 ml of PBS (50 mM, pH 7.0),
1 mM of sodium azide, and 0.5 mM of EDTA. To start the reaction,
0.2 ml of substrate H2O2 (0.15 mM) was added. The mixture was
maintained at 37 °C for 10 min and the rate of NADPH diminution
was measured spectrophotometrically at 340 nm. One unit (U) of
GPx was equivalent to the oxidation of 1 micromole of NADPH per
second.
2.10 | Determination of PON1 activities
Two different activities of PON1 (paraoxonase and arylesterase)
were spectrophotometry dosed in the serum of hamsters. The
used substrates were paraoxon for paraoxonase activity and phe-
nylacetate for arylesterase activity. Each activity assay was per-
formed on two aliquots of each sample in triplicate. To prepare
serum, the blood samples collected from hamsters were centri-
fuged at 4 °C for 10 min at 3000g. A UV spectrophotometry was
used to record, at 25 °C for 5 min, the optical density at 240 nm in
both assays. To calculate paraoxonase and arylesterase activities,
extinction coefficients of 17,000/M/cm and 1310/M/cm were
used, respectively.
2.10.1 | Paraoxonase assay
The capacity to catalyze the conversation reaction of paraoxon into
para-­nitrophenol was evaluated to measure paraoxonase activity.
The generation rate of para-­nitrophenol was determined. Briefly,
serum was 50-­fold diluted in a mixture containing mixed paraoxon
(1.5 mM) served as substrate, Tris hydrochloride (10 mM), sodium
chloride (1 M), and calcium chloride (2 mM) (Bansal et al., 2013). The
results were expressed as μmol of para-­nitrophenol formed per min
per ml of serum.
2.10.2 | Arylesterase assay
This activity was determined according to a previously described
method (Hammadah et al., 2017), with minor modifications. The
phenylacetate hydrolysis rate was determined in 50-­
fold diluted
serum in a tube assay containing substrate phenylacetate (3.4 mM),
calcium chloride (0.9 mM), and Tris hydrochloride (9 mM) at slightly
alkaline (pH 8). The absorbance was monitored at 270 nm for 5 min.
The activity is expressed as μmol of phenylacetate disappearing per
min per ml of serum.
2.11 | Statistical analysis
Values were done as means ± SD (standard deviation). Statistical
analysis is made by ANOVA and Tukey's test using StatView soft-
ware. A p-­value less than 0.05 was considered statistically significant.
3 | R E S U LT S
3.1 | Total phenolic content
The yield of extraction (n = 3) was 17.58 ± 1.93% (w/w). Table 2
shows that Pp had a TPC value of 589.34 ± 43.56 mg CAE/g Pp.
KHOUYA et al.
The chromatographic analysis by HPLC was carried out and pub-
lished previously (Khouya et al., 2020). Rosmarinic acid and querce-
tin are the more abundant phenolic acids in the Pp.
3.2 | Glycemia and insulin levels
The results presented in Table 3 demonstrate that the HFD sig-
nificantly (p < .05) increased blood glucose and plasma insulin
in comparison with the group feeding a normal diet (control).
Compared to the HF group, hamsters in HFD and receiving Pp
had their values of blood glucose (89.33 ± 5.50 mg/dL, p < .05)
and plasma insulin (1.36 ± 0.36 ng/dL, p < .05) statistically
lowered.
3.3 | Lipoprotein profiles
The chromatograms of plasma lipoprotein TC and TG are shown in
Figures 1 and 2. The results showed that the majority of the cho-
lesterol portion in the plasma was in the form of HDL-­C , while the
plasma TG seemed to be distributed among different lipoproteins
in hamsters feeding on the normal (standard) diet. HFD altered the
lipid profile, as evidenced by significant increases in the levels of
all lipoprotein parameters, with a significant increase in very-­low-­
density lipoprotein (VLDL)-­TG and LDL-­C compared to the control.
HFD with Pp significantly increased HDL-­C concentration, which
was higher than the HDL-­C content in two other groups (HF and
control). LDL-­C in the group receiving plant extract was similar to
that in the control.
TA B L E 2 Total phenolic content of the polyphenol-­rich extract
Polyphenol-­rich
extract
Total phenolic content 589.34 ± 43.56 CAE
Note: CAE: mg caffeic acid/g dried extract.
TA B L E 3 Fasting blood glucose and plasma insulin in the three
groups
Insulin
Groups Glucose (mg/dl) (ng/ml)
Control 113.20 ± 6.05 1.91 ± 0.10
HF 117.20 ± 9.52* 2.64 ± 0.70*
HF-­Pp 89.33 ± 5.5* 1.36 ± 0.36*
Notes: The values are the mean ± SD of 10 animals. Control received
a standard diet and was administered orally with distilled water. The
HF group was fed with HFD and administered with distilled water. The
HF-­Pp group was fed with HFD and given a polyphenol-­rich extract
(40 mg/100 g/day) for 63 days. Results are analyzed with ANOVA and
Tukey's test. ns: not significant, *p < .05; HF vs. control. HF-­Pp vs. HF.
| 5 of 11
3.4 | Antioxidant activity in vivo
T. atlanticus Pp's in vivo antioxidant properties were assessed by
measuring plasma MDA levels, and the activity of plasma SOD and
GPx in the three groups (Figure 3).
3.4.1 | MDA
The MDA level in the plasma of the HF group was found to be
statistically higher than that in the normolipidemic group (p < .01,
Figure 3a). Compared to HF, the MDA level was also found to be sta-
tistically lower (p < .01) in the HF-­Pp group (Figure 4a). The plasma
MDA level in HF-­Pp was similar to that in the control.
3.4.2 | Activity of SOD
As shown in Figure 3b, the activity of serum SOD was effectively
decreased (by 45%, p < .001) in HF compared to control. No signifi-
cant difference (p > .05) was noted between HF-­Pp and HF groups.
3.4.3 | Activity of GPx
The results illustrated in Figure 3c indicate that HFD significantly
increased GPx activity (by about 93%) in comparison to control
(p < .001). Supplementing HFD with Pp did not affect GPx activity
when we compared with hamsters feeding HFD alone (p > .05).
3.5 | Paraoxonase and arylesterase activities
As illustrated in Figure 4, the HFD had no significant effect (p < 0.05)
on both paraoxonase and arylesterase activities compared to the
standard diet group. Paraoxonase and arylesterase activities were
significantly (p < .001) higher in the HF-­Pp group by 61% and 33%,
respectively, in comparison to control.
4 | DISCUSSION
Lipid-­rich diets have become one of the most serious public health
threats. They are responsible for diabetes, obesity, and other com-
plications as well as elevated production of atherogenic products
and insulin signaling perturbation (Kumar et al., 2021).
The hamster is a useful model for studying chronic hyperlipid-
emia and its complications because it resembles humans in terms
of lipid metabolism and the development of metabolic disorders in
response to HFD (Sullivan et al., 1993). In this study, we used ham-
sters feeding an HFD as a model for studying how a polyphenol-­rich
extract of T. atlanticus affects hyperlipidemia, oxidative stress, and
insulin resistance.
6 of 11 | KHOUYA et al.

F I G U R E 1 Representative TG profiles of different groups performed by FPLC. Control (a), HF (b), and HF-­Pp (c). The control group was
fed a normal diet and given distilled water orally. The HF group was fed with HFD and received distilled water. HF-­Pp was fed with HFD and
received polyphenol-­rich extract (40 mg/100 g/day) for 63 days

F I G U R E 2 Representative cholesterol profiles of different groups performed by FPLC. Control (a), HF (b), and HF-­Pp (c). The control
group was fed a normal diet and given distilled water orally. The HF group was fed with HFD and received distilled water. HF-­Pp was fed
with HFD and received polyphenol-­rich extract (40 mg/100 g/day) for 63 days

We discovered that feeding hamsters HFD for 63 days resulted
in: (i) a change in plasma lipoprotein profile, primarily manifested
by increased concentrations of VLDL-­TG and LDL-­C , (ii) a change in
the serum antioxidant defense system by modulating antioxidant
enzyme activities and increasing serum oxidant compound (MDA)
concentration, and (iii) the development of insulin resistance, as ev-
idenced by hyperglycemia and elevated plasma insulin levels. These
changes following HFD feeding have been found in many previous
studies using animals receiving HFD, and the current model appears
to be effective in studying hyperlipidemia and its complications and
appropriate in evaluating the effect of T. atlanticus on lipoprotein
profile, oxidative stress, and insulin resistance.
HFD was found to increase lipid levels in the bloodstream (Duan
et al., 2018). Dietary lipids are transported in chylomicrons from the
intestine in the bloodstream in which they are liberated as TG (Ko
et al., 2020). The majority of these TG is mainly taken up by the liver
and promotes the generation of VLDL, which in turn converts into
LDL (Ko et al., 2020). A portion of TG is accumulated in the liver
where it can be degraded via the mitochondrial β-­oxidation. This
leads to an excess electron flow using mitochondrial cytochrome-­c
oxidase and enhances the production of ROS. Excessive circulating
LDL particles and ROS production amplify the generation of oxidized
LDL-­C (Kattoor et al., 2018). The modified LDL-­C is not recognized
by hepatocyte receptors and will be accumulated in macrophages
which transform into foam cells (Kattoor et al., 2018). The latter has
a crucial role in atherosclerosis occurrence and progression (Kattoor
et al., 2018). In addition, elevated circulating fatty acids result in in-
sulin resistance development (Duan et al., 2018). Furthermore, HFD
consumption has been reported to impair the liver–­gut axis, disturb
the release of factors from these organs, and upregulate exosome
phosphatidylcholine, which contributes to insulin resistance (Kumar
et al., 2021).
In this study, a group of HFD-­fed hamsters received a daily
dose of 40 mg/100 g of T. atlanticus Pp. Pp showed beneficial
KHOUYA et al. | 7 of 11

F I G U R E 3 MDA levels and antioxidant enzyme activities in the plasma of the three groups. MDA levels in plasma (a), SOD activity (b),
and GPx activity (c). Control received a standard diet and was administered orally with distilled water. The HF group was fed with HFD and
administered with distilled water. HF-­Pp was fed with HFD and received polyphenol-­rich extract (40 mg/100 g/day) for 63 days. Results are
presented as mean ± SD (n = 10). Statistical analysis was performed by ANOVA and Tukey's test. ns: not significant, *p < .01, **p < .001; HF
vs. control. HF-­Pp vs. HF

F I G U R E 4 Paraoxonase (a) and arylesterase (b) activities of serum PON1 in the three groups. The control group was fed a standard diet
and received distilled water. The HF group was fed a high-­fat diet (HFD) and received distilled water. The HF-­Pp group was fed with HFD
supplemented with polyphenol-­rich extract (40 mg/100 g/day). The results are presented as the mean ± SD (n = 10). Statistical analysis was
performed by ANOVA and Tukey's test. ns: not significant, *p < .001; HF vs. control. HF-­Pp vs. HF

effects on studied parameters. Hamsters receiving Pp had a lipo-
protein profile similar to that of the group feeding a normal diet,
with an important reduction in VLDL-­TG and LDL-­C in compar-
ison with the group feeding HFD alone. This is consistent with
available studies, which have shown that extracts of T. atlanticus
ameliorated chronic hyperlipidemia in HFD-­fed mice and allevi-
ated the alteration in the lipoprotein profile induced by Triton in
golden hamsters (Ramchoun, Khouya, Harnafi, Alem, et al., 2020;
Ramchoun, Khouya, Harnafi, Amrani, et al., 2020). The Pp of T.
atlanticus has been found to decrease serum cholesterol by down-
regulating HMG reductase and to decrease liver and plasma TG
in hamsters feeding on HFD (Ramchoun, Khouya, Alibrahim,
Hmidani, et al., 2020). The inhibitory effect of T. atlanticus on cho-
lesterol biosynthesis and its antihypertriglyceridemia effect could
explain the decrease in LDL-­C level and ameliorated lipoprotein
profile observed in this study.
Hyperlipidemia may result in oxidative conditions in the body
(Lian et al., 2020). The ameliorated lipid profile observed in the group
receiving Pp was accompanied by an improvement in antioxidant
status. The amount of MDA was significantly lower in the Pp-­treated
group. MDA is a highly toxic molecule and is considered one of the
final products of peroxidation (Wadhwa et al., 2019). This aldehyde
can interact with proteins and DNA and induce potentially muta-
genic and atherogenic effects (Wadhwa et al., 2019). Low serum
8 of 11 | KHOUYA et al.
MDA levels in the treated group signify that Pp reduced lipid per-
oxidation and, thereby, can play antiatherogenic and antimutagenic
propriety.
Aside from the amount of serum lipid oxidation products, the
measurement of antioxidant enzyme activity is known as an indi-
cator to evaluate the antioxidant status and oxidative damage. In
this context, we evaluated the enzymatic activity of serum SOD and
GPx. SOD is the only antioxidant enzyme that can neutralize super-
oxide anion. Many studies show that SOD is involved in the regula-
tion of several physiological functions such as lipid metabolism and
inflammation as well as protecting cells from oxidative stress (Islam
et al., 2021). GPx, a selenium-­dependent enzyme, removes H2O2 and
reduces organic peroxides to H2O by the oxidation of reduced gluta-
thione (Mahl et al., 2015).
We revealed that HFD caused a marked increase in SOD and GPx
activities. This is consistent with previous work, which reported an in-
creased activity of several antioxidant enzymes to limit oxidative stress
induced by HFD in animal models (Küçükkurt et al., 2010). Moreover,
the gene promoter region of many antioxidant enzymes contains nu-
clear factor kappa B (NF-­κB)-­binding sites (Lingappan, 2018). This nu-
clear factor has been reported as a link between overproduced ROS
and activation of antioxidant defense. Most studies have found that
the activation of NF-­κB upregulates GPx and SOD. ROS have been
shown to act as signals and activate NF-­κB, which results in an upreg-
ulation of antioxidant enzymes (Lingappan, 2018).
In our study, supplementation with Pp did not reverse the effect
of HFD on SOD and GPx. The in vivo antioxidant effect of Pp may be
attributed to other mechanisms.
In this study and other previous studies reporting on hyperlipid-
emic animals, extracts from T. atlanticus were shown to slightly in-
crease HDL-­C levels (Ramchoun, Khouya, Harnafi, Alem, et al., 2020;
Ramchoun, Khouya, Harnafi, Amrani, et al., 2020). Many findings re-
veal that HDL levels are proportional to diminished cardiovascular
risk (Lin et al., 2018; Nagao et al., 2018). However, HDL-­C raising
strategies have not been proven to have a protective effect against
vascular events. Independently of their plasma levels, HDL can be-
come dysfunctional due to compositional changes and modifications
of associated enzymes (Bonizzi et al., 2021). HDL dysfunction has
been found in patients with cardiovascular disease, insulin resistance,
or hyperlipidemic subjects. Measuring HDL function is considered a
good tool to predict its antiatherogenic effects (Bonizzi et al., 2021).
The function of an HDL-­associated enzyme PON1 was evaluated in
the present study. PON1 is found to stabilize and protect HDL and
LDL particles against oxidative modification (Chistiakov et al., 2017).
Here, we revealed that HFD seemed to have no significant effect
on arylesterase and paraoxonase activities. Even when compared to
group feeding a normal diet, combined administration of HFD with
Pp effectively raised arylesterase and paraoxonase activities. Similar
results have been revealed in previous studies using polyphenol-­rich
extracts (Martini et al., 2017). The hepatic expression of PON1 was
previously shown to be upregulated by polyphenols, such as quer-
cetin, resveratrol, and ellagitannins (Ibrahim et al., 2021; Khateeb
et al., 2010; Tabatabaie et al., 2020). The possible mechanisms of the
stimulator effect of polyphenols on PON1 remain largely unknown,
but the nuclear receptor peroxisome proliferator-­activated recep-
tor γ (PPAR γ) is suggested as a molecular target for polyphenols
(Khateeb et al., 2010).
Increased arylesterase and paraoxonase activities in the Pp-­
treated group could be related to increased HDL-­C levels as well
as upregulated PON1 expression independently of HDL-­C levels.
Due to its antioxidant and antiatherogenic properties, PON1 ac-
tivity modulation could be a useful strategy for preventing several
diseases, such as heart and vessel diseases, insulin resistance, and
others (Moya & Máñez, 2018).
PON1 has been found to ameliorate the ability of HDL to
transport cholesterol from peripheral tissues to the liver (Ikhlef
et al., 2017), which may explain the cholesterol-­lowering effect fol-
lowing Pp administration observed in previous studies (Ramchoun
et al., 2012; Ramchoun, Khouya, Harnafi, Alem, et al., 2020).
Previous studies have reported interesting in vivo anti-­
inflammatory effects of Pp in animal models (Khouya et al., 2015,
2020, 2021). This may be due to the actions of polyphenols con-
tained in Pp on PON1 activity, as enhanced PON1 activity protects
against inflammatory and atherogenic processes. A plant extract
contains a wide range of phytochemicals including polyphenols,
which can act synergically and additively to exert their effects
(Martini et al., 2017).
The current study showed that HFD intake markedly raised
blood glucose and plasma insulin levels as a consequence of insu-
lin resistance. These alterations were significantly restored by the
combined administration of HFD with Pp compared to HFD-­fed
hamsters. Thus, Pp suggested improving insulin resistance and re-
storing glucose levels in HFD-­fed hamsters, which complies with
previous reports using polyphenol-­rich plant extracts (Williamson &
Sheedy, 2020). Oxidative stress has been found to have a key role
in insulin resistance development (Yaribeygi et al., 2020). ROS may
perturb intracellular signaling and contribute to insulin resistance
(Yaribeygi et al., 2020). Pp administration significantly ameliorated
oxidative stress, evidenced by reduced plasma MDA levels and in-
creased PON1 activity, which may result in the enhancement of in-
sulin signaling.
The findings from the current and previous studies indicate that
polyphenol-­rich T. atlanticus extracts may alleviate cardiovascular
and metabolic diseases by reducing plasma cholesterol and LDL-­C ,
liver and plasma TG, and rising HDL-­C levels and their associated
PON1, inhibiting lipid peroxidation, restoring glucose and insulin ho-
meostasis, and due to their in vivo and in vitro actions on oxidative
stress and inflammation. T. atlanticus is a rich source of polyphenols
and flavonoids, which are probably responsible for the observed
pharmacological benefits.
5 | CO N C LU S I O N
Our work showed that the Pp of T. atlanticus alleviated dyslipidemia,
decreased serum MDA levels, enhanced both arylesterase and
KHOUYA et al.
paraoxonase activities of PON1, and improved insulin resistance
induced by HFD in hamsters. Polyphenols from T. atlanticus are sug-
gested to be effective in ameliorating metabolic diseases linked to
the consumption of high-­fat diets.
AU T H O R C O N T R I B U T I O N S
Tarik Khouya: Conceptualization; software; validation; writing –­
original draft; writing –­review and editing. Mhamed Ramchoun:
Investigation; methodology; resources; validation; writing –­review
and editing. Abdelbassat Hmidani: Software; validation; writing –­
original draft. Souliman Amrani: Conceptualization; methodology;
validation. Mohamed Benlyas: Formal analysis; investigation; valida-
tion. Fatima Kasbi Chadli: Data curation; methodology; validation.
Khadija Ouguerram: Methodology; supervision; validation. Chakib
Alem: Conceptualization; supervision; validation; writing –­ review
and editing.
AC K N OW L E D G M E N T
The authors thank Dr Ibn Tatou for plant authentication.
C O N FL I C T O F I N T E R E S T
There is no conflict of interest.
DATA AVA I L A B I L I T Y S TAT E M E N T
All the data used is available from the corresponding author.
E T H I C S A P P R OVA L
The study involved 30 hamsters and was conducted according to
animal welfare laws and guidelines. Animal protocols were approved
by the University of the local Animal Used and Care Advisory
Committee (Bretagne-­Pays de la Loire committee) and conform to
Directive 2010/63/EU.
ORCID
Tarik Khouya https://orcid.org/0000-0002-9122-7766
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