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Evaluation of Possible Antioxidant, Anti-Hyperglycaemic, Anti-Alzheimer

and Anti-Inflammatory Effects of Teucrium polium Aerial Parts (Lamiaceae)

Article in Life · October 2022

DOI: 10.3390/life12101579

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 life

Article
Evaluation of Possible Antioxidant, Anti-Hyperglycaemic,
Anti-Alzheimer and Anti-Inflammatory Effects of
Teucrium polium Aerial Parts (Lamiaceae)
Naima Benchikha 1 , Mohammed Messaoudi 1,2 , Imane Larkem 3 , Hamza Ouakouak 1 , Abdelkrim Rebiai 1 ,
Siham Boubekeur 4 , Mohamed Amine Ferhat 5 , Adel Benarfa 6 , Samir Begaa 2 , Mokhtar Benmohamed 7 ,
Diena M. Almasri 8 , Rawan H. Hareeri 9 and Fadia S. Youssef 10, *

1 Laboratory of Applied Chemistry and Environment (LCAE), Chemistry Department, University of Hamma
Lakhdar El-Oued, B.P.789, El-Oued 39000, Algeria
2 Nuclear Research Centre of Birine, P.O. Box 180, Ain Oussera, Djelfa 17200, Algeria
3 Laboratory of Diversity of Ecosystems and Dynamics of Agricultural Production Systems in Arid Zones,
Department of Agronomy, Faculty of Nature and Life Science, Biskra University, Biskra 07000, Algeria
4 Research and Development Centre RDC-SAIDAL, 35Benyoucef Khattab Avenue, Mohammadia, El-Harrah,
Algiers 16000, Algeria
5 Ecole Normale Supérieure (ENS), P.O. Box 92, Vieux-Kouba, Alger 16308, Algeria
6 Centre de Recherche Scientifique Et Technique en Analyses Physico-Chimiques (CRAPC)-PTAPC,
P.O. Box 0354, Laghouat 03000, Algeria
7 Laboratory of Fundamental Sciences, University Amar Télidji of Laghouat, P.O. Box 37G, Road of Ghardaïa,
Laghouat 03000, Algeria
8 Department of Pharmacy Practice, Faculty of Pharmacy, King Abdulaziz University,
Jeddah 21589, Saudi Arabia
9 Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University,
Citation: Benchikha, N.; Messaoudi,
Jeddah 21589, Saudi Arabia
M.; Larkem, I.; Ouakouak, H.; Rebiai,
10 Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Abbasia, Cairo 11566, Egypt
A.; Boubekeur, S.; Ferhat, M.A.;
* Correspondence: [email protected]
Benarfa, A.; Begaa, S.; Benmohamed,
M.; et al. Evaluation of Possible
Abstract: Teucrium polium L. is commonly used in folk medicine to treat hypertension and diabetes
Antioxidant, Anti-Hyperglycaemic,
Anti-Alzheimer and Anti-
and to heal wounds. The present work aimed to evaluate the different biological activities of T. polium
Inflammatory Effects of Teucrium hydroalcoholic extract, its total phenol and flavonoid content, and its mineral elements. Results showed
polium Aerial Parts (Lamiaceae). Life that T. polium extract showed significant antioxidant potential in 2-diphenyl-1-picrylhydrazyl (DPPH)
2022, 12, 1579. https://doi.org/ assay with IC50 equal to 8.68 µg/mL but with moderate activity in galvinoxyl assay with IC50 of
10.3390/life12101579 21.82 µg/mL and mild activity in the β-carotene assay. It also showed a pronounced anti-hyperglycemic
activity using α-amylase inhibitory assay (IC50 = 111.68 µg/mL) and exceeds that of acarbose.
Academic Editor: Stefania Lamponi
T. polium showed excellent activity against acetylcholinesterase (AChE) and butyrylcholinesterase
Received: 12 September 2022 (BChE) with IC50 values of 28.69 and 4.93 µg/mL, respectively, postulating its promising anti-Alzheimer
Accepted: 8 October 2022
potential. The plant extract exhibited a strong anti-inflammatory effect with Bovine Serum Albumin
Published: 11 October 2022
(BSA) denaturation inhibitory potential estimated by 97.53% at 2 mg/mL, which was further con-
Publisher’s Note: MDPI stays neutral firmed by the in vivo carrageen-induced edema model. The extract revealed its richness in flavonoids
with regard to jurisdictional claims in and phenols, evidenced by its polyphenols content (36.35 ± 0.294 µg GAE/mg) and flavonoids
published maps and institutional affil- (24.30 ± 0.44 µg QE/mg). It is rich in minerals necessary for human health, such as calcium, potas-
iations. sium, iron, sodium, magnesium, manganese and zinc. Molecular docking performed for previously
identified compounds on human α-amylase, 5-lipoxygenase (5-LOX) and acetylcholine esterase con-
firmed the results. Thus, it can be concluded that T. polium can be a good candidate for alleviating
many health-debilitating problems and can be highly beneficial in the pharmaceutical industry and
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
medical research.
This article is an open access article
distributed under the terms and Keywords: Teucrium polium L.; antioxidant; anti-hyperglycemic; anti-Alzheimer; anti-inflammatory;
conditions of the Creative Commons molecular docking; drug discovery; health care
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Life 2022, 12, 1579. https://doi.org/10.3390/life12101579 https://www.mdpi.com/journal/life

Life 2022, 12, 1579 2 of 23

1. Introduction
Oxidative stress has been recognized as the main cause of many life-threatening
diseases, comprising atherosclerosis, cardiovascular diseases, diabetes, obesity, cancer, and
many inflammatory conditions [1,2]. The unregulated inflammatory responses further
worsen the previously mentioned disorders that could be managed via the consumption of
steroidal or non-steroid anti-inflammatory and immunosuppressant drugs. However, they
trigger a plethora of side effects [3,4]. In addition, diabetes mellitus and Alzheimer’s disease
(AD) are among the most debilitating disorders that greatly influence patients’ capabilities
causing a significant reduction in their activities and affecting their well-being [5].
Naturally occurring antioxidants of plant origin are highly popular for combatting
oxidative stress and counteracting inflammation and its associated disorders, owing to their
richness with bioactive secondary metabolites [6–9]. Furthermore, they have the potential
to effectively inhibit oxidative stress that is associated with diseases such as diabetes
and neurodegenerative disorders [10]. As a result, they are highly welcomed by a large
category of patients all over the globe and, thus, can be used as an alternative to synthetic
drugs due to their lower adverse effects and price compared to synthetic ones [11,12]. The
pronounced anti-inflammatory potential of medicinal plants is mainly attributed to their
richness in natural antioxidants, such as flavonoids, polyphenols, tocopherols, carotenoids,
and ascorbic acid [1,13].
Genus Teucrium belonging to the family Lamiaceae includes about 300 species greatly
spread throughout North Africa, Europe and Asian temperate regions [14], where its
different species showed many activities [15,16]. Teucrium polium L. is widely used in
traditional medicine to treat hypertension and diabetes or as a wound-healing agent [13].
T. polium is a deciduous shrub native to the Western Mediterranean region [17]. It showed
many biological activities, such as anti-inflammatory, antiviral, antifungal, antibacterial,
cytotoxic, antioxidant, hypoglycemic, hypolipidemic, hepatoprotective, analgesic, antiulcer
effects, in addition to anticonvulsant potential. These activities are highly attributed to
plants’ bioactive secondary constituents, such as phenylethanoid glycosides, flavonoids,
diterpenes, iridoids and essential oil [13,14,17].
In this study, we aimed to evaluate the antioxidant, anti-hyperglycaemic, anti-Alzheimer
and anti-inflammatory effects of Teucrium polium hydroalcoholic extract from the aerial parts.
The antioxidant was assessed using 2-diphenyl-1-picrylhydrazyl (DPPH), β-carotene and Galvi-
noxyl radical (GOR) assays, whereas the anti-hyperglycemic was determined in vitro by the
α-amylase inhibition method. The anti-inflammatory effect was determined in vitro by inhibit-
ing denaturation of BSA (Bovine Serum Albumin) and in vivo by inhibiting mouse paw edema
induced by carrageenan. However, the anti-Alzheimer activity of the extract of T. polium aerial
parts was assessed in vitro via the determination of the anti-cholinesterase activity carried out
by the acetylcholinesterase inhibitor method (AChE) and butyrylcholinesterase (BChE). Total
phenol and flavonoid content, as well as mineral contents, were evaluated for the first time.
Moreover, the correlation between the studied activities and its major previously identified
metabolites was determined in silico using molecular docking within the active sites of human
α-amylase (HA), acetylcholine esterase and 5-lipoxygenase using Discovery Studio 4.5 (Accelrys
Inc., San Diego, CA, USA) with C-docker protocol to further consolidate the obtained results.
This work is part of an overall program in our laboratory to use nuclear analytical techniques
for studying natural food samples relevant to human health and nutrition.

2. Materials and Methods

2.1. Reagents and Materials
Folin-Ciocalteu (FCR), sodium carbonate (Na2 CO3 ),2,2-diphenyl-1-picrylhydrazyl
(DPPH), 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ABTS, α-Tocopherol
(Vitamin E), 2,6-ditert- butyl-4-methylphenol (BHT), gallic acid, quercetin, and AlCl3
were purchased from Sigma (Sigma-Aldrich, Germany). All the organic solvents and other
chemicals used in the present study were of analytical grade and were obtained from
Sigma-Aldrich (Sigma Aldrich, Germany).
Life 2022, 12, 1579 3 of 23

2.2. Plant Material

The aerial parts of Teucrium polium L. (Lamiaceae family) were collected on June 2018
from the El-Guetfa region, Msila, located in the semi-arid region of Algeria (35◦ 440 26 N
and 3◦ 230 05 E). The plant material was identified taxonomically based on the identification
methods described by Quezel and Santa [18] and by one of the authors, H.O., and Professor
A. Hassani (ENS-kouba). A voucher sample was stored in the Chemistry Department,
University of Hamma Lakhdar El-Oued, Algeria, under the code (RO-050). The aerial parts
were cleaned and dried. Dry aerial parts were pounded with an electric blender and kept
wrapped in paper until use.

2.3. Preparation of the Plant Hydroalcoholic Extract

The aerial parts of T. polium L. samples were washed several times with deionized
water and dried for two weeks at room temperature. The dried samples were ground to a
fine powder (particle size fraction of <200 µm) using an agate mortar and pestle. After that,
20 g of plant powder was soaked in a solvent fraction of ethanol/water (80/20) (%) for
24 h, then filtered and dried in a rotary vapor under reduced pressure at 45 ◦ C. The
extraction yield was calculated and found to be 20.45%

2.4. Determination of the Biological Activity of T. polium Hydroalcoholic Extract

2.4.1. Evaluation of Possible Antioxidant Activity In Vitro
The antioxidant activity of T. polium L. hydroalcoholic extract was achieved us-
ing several analytical tests distinguished by their mechanism of reaction 2-diphenyl-1-
picrylhydrazyl (DPPH), β-carotene and galvinoxyl radical (GOR). The reaction was initi-
ated by mixing 0.5 mL of hydroalcoholic extract with 1 mL of DPPH (0.2 mM), β-carotene
(0.5 mM) and GOR (0.1 mM) using the protocols described in references [19,20]. The mix-
tures were left in the dark at room temperature for approximately 30 min; after that, the
absorbance was measured at λmax : 596, 490 and 428 nm for the mentioned tests, respectively,
against a blank using a UV-vis spectrophotometer apparatus (Shimadzu UV-2450, Japan).
The inhibition percentage was calculated as follows: I% = ((A0 − A1 )/A0 ) × 100, where A0
is the absorbance of DPPH, β-carotene and GOR solutions without extracts, while A1 were
the absorbance of the sample. The activity results for all tests were expressed as IC50 (half
maximal inhibitory concentration) values, which have been calculated (using Microsoft
Excel 2016) from an antiradical linear graph formed by the X axis, which represented extract
concentration, and the Y axis, which represented their relative inhibition percentage. In
addition, the antioxidant activity results were compared with three standards, BHA, BHT
and vitamin, as represented in Table 1.

Table 1. In vitro antioxidant activity of T. polium hydroalcoholic extract (TPL) using DPPH, GOR and
β carotene assays expressed in terms of IC50 (µg/mL).

Samples DPPH GOR β Carotene

TPL 8.68 ± 0.33 21.82 ± 0.19 165.41 ± 1.57
BHT 12.99 ± 0.41 3.32 ± 0,18 0.91 ± 0.01
BHA 6.14 ± 0.41 5.38 ± 0,06 1.05 ± 0.03
Vitamin E 15.81 ± 0.54 - -
Values were expressed as means ± SD (n = 3) for three parallel measurements.

2.4.2. Evaluation of Possible Anti-Hyperglycemic Activity In Vitro

The anti-hyperglycemic activity of the T. polium aerial parts hydroalcoholic extract was
tested using the α-amylase inhibition method according to the Zengin method with some
modifications [21]. On a 96-well microplate reader (Perkin Elmer EnSpire, Singapore), a
volume of 25 µL of the hydroalcoholic extract at different concentrations (62.5, 125, 250, 500
and 4000 with an increment of µg/mL) was mixed with 50 µL of 1U α-amylase solution
and then incubated for 10 min at 37 ◦ C. Then 50 µL of starch (0.1%) was added. The
mixture was incubated again for 10 min at 37 ◦ C. After incubation, 25 µL of hydrochloric
Life 2022, 12, 1579 4 of 23

acid (1 M) (prepared by diluted 4.17 mL of pure HCl with 45.83 mL distilled water) and
100 µL of iodine-potassium iodide solution was added. The absorbance was read at 630 nm.
Acarbose was used as a standard. The following formula was used to calculate α-amylase
inhibition percentage: I% = 1 − [(Ac − Ae ) − (As − Ab )/(Ac − Ae )]
Ac = Absorbance [Starch + IKI + HCL + Extract solvent volume + Enzyme
buffer volume]
Ae = Absorbance [Enzyme + Starch + IKI + HCl + Vol. extract solvent]
As = Absorbance [Enzyme + Extract + Starch + IKI + HCl]
Ab = Absorbance [Extract + IKI + 125 µL of buffer]

2.4.3. Evaluation of Possible Anti-Alzheimer Activity In Vitro

The anti-Alzheimer activity of the hydroalcoholic extract of T. polium aerial parts was
assessed in vitro via the determination of the anti-cholinesterase activity carried out by
the method of acetylcholinesterase inhibitor (AChE) and butyrylcholinesterase (BChE).
This was measured using the spectrophotometric method previously reported by Ellman
with slight modifications [22]. AChE from electric eel and BChE from horse serum were
used, while acetylthiocholine iodide (ACI) and butyrylthiocholine chloride (BuCi) were
used as reaction substrates. DTNB solution (5,50 -Dithiobis (2-nitro-benzoic acid)) was
used to measure cholinesterase activity. Briefly, 150 µL of 100 mM sodium phosphate
buffer (pH = 8), 10 µL of the sample solution (4 mg/mL) dissolved in methanol at different
concentrations, and 20 µL of AchE or BchE solution prepared in a buffer (pH = 8) were
mixed and incubated for 15 min at 25 ◦ C. Then 10 µL of D-TNB (0.5 mM) were added.
The reaction was then initiated by adding 10 µL of acetylthiocholine iodide (0.71 mM)
or 10 µL of butyrylthiocholine chloride (0.2 mM). The hydrolysis of these substrates was
monitored spectrophotometrically at a wavelength of 412 nm using a 96-well microplate
reader (Perkin Elmer EnSpire, Singapore) by forming a yellow 5-thio-2-nitrobenzoate anion
resulting from the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis
of acetylthiocholine iodide or butyrylthiocholine chloride, respectively. Galanthamine was
used as a reference compound, and the results were given as percent inhibition against
concentrations of 25, 50, 100 and 200 µg/mL; the IC50 was also given. The percent inhibition
of BChE and AChE was determined using the following formula: I% = [(E − S)/E] ∗ 100,
where E: the activity of the enzyme without extract, and S: the activity of the enzyme with
the extract.

2.4.4. Evaluation of Possible Anti-Inflammatory Activity In Vitro

In vitro anti-inflammatory activity was determined for the hydroalcoholic extract of
T. polium aerial parts using the inhibiting denaturation of BSA (Bovine Serum Albumin)
following the method previously reported by Kandikattu et al. [23], with slight modifications.
It relied upon the inhibition of the denaturation of BSA caused by heat (72 ◦ C). Briefly, 1 mL
of each concentration of extract or standard (Diclofenac sodium) was added to 1 mL of 0.2%
BSA solution prepared in tris-HCl (pH = 6.6). The solutions were incubated at 37 ◦ C for
15 min in an oven, then in a water bath at 72 ◦ C for 5 min. After cooling, the turbidity was
measured at 660 nm in a UV-visible spectrophotometer. For each concentration of extract, a
blank is prepared in 1 mL of hydroalcoholic extract and 1 mL of tris-HCl (the purpose of this
blank is to subtract the absorbance of the extract from the results obtained). The protective
effect of samples against the denaturation of BSA was presented as inhibition percentages
calculated using the following formula: I% = [(Ac − As)/As] ∗ 100, where I%: the inhibition
percentage, AC : absorbance of the control and AS : absorbance of the tested sample.

2.4.5. Evaluation of the Anti-Inflammatory Activity In Vivo

Experimental Animals
The anti-inflammatory activity was performed in vivo using albino mice of Swiss
strain from the Pasteur Institute (Algiers). Male mice were used with weights ranging
from 20 to 22 g. The laboratory animals were housed in plastic cages at room temperature
Life 2022, 12, 1579 5 of 23

(25 ◦ C) and exposed to light for 12 h per day. During the acclimatization period (a week
before being used in the various experiments), the mice had free access to water and food
(croquettes from the Animal Feed Production Company, Bouzareah, Alger).

Experimental Protocol
The anti-inflammatory activity was assessed by inhibiting mouse paw edema induced
by carrageenan following the protocol previously described by Colot. [24]. The principle of
this study consists of injecting carrageenin under the plantar fascia of the left paw of the
mouse to cause an inflammatory reaction, which can be reduced by an anti-inflammatory
product. This study allows the comparison of plantar edema after administration of equal
doses of the anti-inflammatory product to be tested (the hydroalcoholic extract of the plant
at 10%) and the corresponding reference product (Diclofenac sodium at 10 mg/kg). The
experiment was carried out as follows:
At time T0 , the mice were divided randomly into 3 batches; each batch contained
5 mice that were made up randomly. In the control group: each mouse received 0.5 mL of
physiological water. Each mouse received a reference anti-inflammatory drug, Diclofenac,
in the standard group at 10 mg/kg. The treated group received the test solution, where
each mouse received 0.5 mL of the plant extract at 10%, where 5 g were suspended in
50 mL H2 O to form a 10% tested solution. Before starting the experiments, the mice fasted
for 16 h, weighed and then were administered intragastrically (gavage) for the three batches
of these suspensions, namely, control solution, reference and extract of the plant. Then half
an hour after dosing, mice from the three batches received 0.025 mL of 1% carrageenan
under the plantar fascia of the left hind paw of the mouse. Then four hours after this
operation, the animals were sacrificed by cervical dislocation, the hind legs were cut at
the height of the joint and then weighed on an analytical balance. The anti-inflammatory
activity was calculated as a percentage reduction in edema in the treated mice compared to
the control according to the following formula:

I% of edema = ((PG − PD) control mouse − (PG − PD) treated mouse )/((PG − PD)control mouse )

where PD = right leg weight, and PG = left leg weight

2.5. Determination of the Total Content of Phenol and Flavonoids

Determination of the total content of phenol and flavonoids was obtained by a spec-
trophotometric method employing the Singleton–Rossi method with the Folin–Ciocalteu
reagent at wavelength λ = 765 nm, using a gallic acid graph as reference for the total phenol
content, whereas quercetin was used as a reference for the total flavonoid content and at
the wavelength λ = 420 nm.

2.5.1. Estimation of Total Phenol Content (TPC)

The total phenol content was determined using the Folin–Ciocalteu reagent [25],
according to a microplate assay method described by Müller [26]. The FCR reagent,
consisting of a mixture of phosphotungstic acid (H3PW12 O40 ) and phosphomolybdic acid
(H3PMo12O40), was reduced, during the oxidation of phenols, to a mixture of tungsten
oxides (W8 O23 ) and molybdenum (Mo8 O23 ). The blue coloration is proportional to the total
phenol content and has a maximum absorption of around 750–765 nm. A volume of 20 µL
of the samples was added to 100 µL of Folin–Ciocalteu reagent diluted to (1:10) and 75 µL
of sodium carbonate (Na2 CO3 ) (7.5%). After 2 h of incubation at room temperature and in
the dark, the absorbance of different intensities of the resulting blue color was measured at
765 nm. A blank was prepared in the same way, replacing the extract with the solvent used
(ethanol/water). Gallic acid with concentrations ranging from 0 to 200 µg/mL was used
to construct the calibration curve as a standard. The results were expressed in µg of gallic
acid equivalent (GAE) by milligram of extract (µg EAG/mg).
Life 2022, 12, 1579 6 of 23

2.5.2. Estimation of Total Flavonoid Content (TFC)

The analysis of flavonoids in the hydroalcoholic extracts is based on the formation
of a complex between Al+3 and the flavonoids. Topçu method [27] was used with some
modifications in a 96-well microplate to determine the total flavonoid content. In total,
50 µL of the extract was mixed with 130 µL of methanol, 10 µL of potassium acetate and
10 µL of aluminum nitrate. After 40 min of incubation in the dark and at room temperature,
the absorbance was read at 415 nm. The concentration of flavonoids in the extracts was
calculated from a calibration curve established with quercetin at different concentrations
ranging from 0 to 40 µg/mL. The results were expressed as equivalent micrograms of
quercetin per milligram of extract (µg EQ/mg of extract).

2.6. Determination of Mineral Elements

Instrumental Neutron Activation Analysis (INAA) and inductively coupled plasma-
optical emission spectrometry (ICP-OES) at Es-Salam Research reactor, Algeria, were used
to determine the concentrations of the elements.

2.6.1. Determination of Mineral Elements by the INAA Technique

In the INAA technique, three samples of each collected charge weighing about 200 mg
were stored in pre-cleaned polyethylene capped bottles. In addition, certified reference
materials, such as NIST-SRM 1573a (tomato leaves) from the National Institute of Standard
and Technology (NIST) and GBW 07605 (National Research Center for CRM. Langfang,
China), were used. All the samples and standards were packed and irradiated together
for 6 h at a thermal neutron flux of 4.5 × 1013 cm−2 s−1 . After appropriate cooling, the
irradiated samples and the standard were measured at different cooling times using a
coaxial HPGe detector with the following characteristics: relative efficiency: 35%, FWHM
1.8 keV for the 1332.5 keV γ-peak of 60 Co. In the end, the concentrations of elements
(major and trace elements) were determined using the equation of INAA given by the
following [28]. 

Np /tc /DC a (ρW )s
ρ(µg/g) = 

Np /tc /DC s Wa
The subscripts a and s refer to the sample and the standard, respectively, Np is the
net photo-peak counts, W is the sample mass, D = [exp (−λ td )] is the decay factor, and
C = ([1 − exp (−λ tm )]/λ tm ) is the counting factor, λ decay constant, tc , td and tm counting,
decay and measurement times, respectively.
To check the accuracy of the analytical method, the parameter of the U-score test was
determined. It was calculated according to the following equation;

|X − XRef |
Uscore = q Lab
µ2Lab + σ2Ref

XLab , µLab , XRef and σRef refer to the laboratory results, the standard deviation, and
the recommended and standard uncertainties, respectively. The laboratory performance
was evaluated as satisfactory if Uscore ≤ 1, and unsatisfactory for Uscore < 1; the result and
certified value were not in agreement [29].

2.6.2. Determination of Mineral Elements by ICP-OES Technique

Determination of the mineral elements by the ICP-OES technique was performed by
putting three samples of each collected charge weighing about 500 mg that were digested
following the protocol previously adopted by the authors [30,31] after the full dissolution
of the sample and allowing the solution to be homogenous by settling.
Life 2022, 12, 1579 7 of 23

2.7. In Silico Studies

2.7.1. Molecular Docking Validation Studies using Re-Docking and Superimposition
Validation of the docking experiments was achieved by comparing the alignments
of the most stable docking poses of the lead compound together with the lead conformer
co-crystallized with the respective enzyme from pdb. The value of RMSD (Root Mean
Square Deviation) was determined to confirm the validity of the docking experiment and
to show the ability to predict the binding affinity of new ligands.

2.7.2. Molecular Docking

Molecular docking was performed for previously identified compounds by HPLC-
UV-MS analysis conducted on Algerian T. polium aerial parts extract [14] using Discovery
Studio 4.5 (Accelrys Inc., San Diego, CA, USA) with C-docker protocol. This was carried
on human α-amylase (HA) (PDB ID 3BAY; 1.99 Å), 5-lipoxygenase (5-LOX) (PDB ID 6N2W,
2.71 Å) and acetylcholine esterase (PDB ID: 4EY7; 2.35 Å) obtained from the protein data
bank (www.pdb.org) following what was previously reported [32,33]. This was compared
with the reference drugs, namely acarbose, nordihydroguaiaretic acid and donepezil,
which are co-crystalized with the previously mentioned enzymes, respectively. This was
briefly performed in several steps, starting with the preparation of the structure of each
enzyme by removing water molecules, followed by the addition of hydrogen atoms and
then cleaning the protein structure from unwanted interactions. CHARMm was used
as the forcefield, whereas MMFF94 was consumed for the calculation of partial charge
calculation that was consequently accompanied by minimizing the added hydrogen in
2000 steps. Determination of the binding center was accomplished depending on the
data reported approaching the catalytic domain of the targeted protein and the sphere
site for the co-crystalized ligand. ChemDraw 13.0 was utilized to draw the 2D structures
of the compounds that subsequently kept as pdb files. The default protocol for ligand
preparation was adopted for the preparation of the 3D structures of the compounds for
docking experiments. Molecular docking was then performed for the prepared structure
inside the binding site spheres of the energy-minimized protein where the CHARMm force
field was selected and the C-docker binding energy (∆G) was calculated using a distance-
dependent dielectric implicit solvation model for the best docking poses. Calculation of
(∆G) in Kcal/mol was performed by employing the following equation:
∆Gbinding = Ecomplex − (Eprotein + E ligand ) where;
∆Gbinding : The ligand–protein interaction binding energy,
Ecomplex : The potential energy for the complex of protein bound with the ligand,
Eprotein: The protein potential energy alone
Eligand : The ligand potential energy alone

2.7.3. ADMET and Drug Likeness Prediction

ADMET (absorption, distribution, metabolism, excretion and toxicity) determination
for the prediction of the pharmacokinetic, pharmacodynamic and toxicity properties and
drug likeness was performed on previously identified compounds by HPLC-UV-MS anal-
ysis, conducted on Algerian T. polium aerial parts extract using Biovia Discovery Studio
software (Accelrys Inc., San Diego, CA, USA). Plasma protein binding prediction (PPB),
human intestinal absorption, aqueous solubility, blood–brain barrier penetration (BBB),
and cytochrome P450 (2D6) and hepatotoxicity level were chosen as ADMET parameters.
However, ALogP, molecular weight (M.W.), number of hydrogen bond acceptors (HBA),
number of hydrogen bonds donor (HBD), number of rings, number of aromatic rings and
number of rotatable bonds were selected as drug likeness descriptors [34].

2.8. Statistical Analysis

Statistical analysis for in vitro experiments was performed by taking the means ± SD
(n = 3) of three parallel measurements. For in vivo experiments, it was performed using
student t-test and one way analysis (ANOVA), and data were expressed as mean ± SD.
Life 2022, 12, 1579 8 of 23

Graphs were constructed by GraphPad Prism version 5 software (GraphPad Software, Inc.
La Jolla, CA, USA)

3. Results
3.1. Determination of the Biological Activity of T. polium Hydroalcoholic Extract
3.1.1. Evaluation of Possible Antioxidant Activity In Vitro
The antioxidant activity of T. polium hydroalcoholic extract was estimated using
DPPH: DPPH, GOR and β carotene assays. The results presented in Table 1 showed
that T. polium hydroalcoholic extract showed moderate activity in galvinoxyl with IC50 of
21.82 µg/mL; it exhibited poor activity in β carotene assay, displaying an IC50 value of
165.41 µg/mL compared to the standards, butylated hydroxyanisole (BHA) and tert-butyl-
1-hydroxytoluene (BHT). In contrast, it showed a significant antioxidant potential in DPPH
assay with IC50 equals 8.68 µg/mL approaching that of BHA (IC50 = 6.14 µg/mL) and
exceeding that of BHT (IC50 = 12.99) and vitamin E (α-Tocophérol (IC50 = 15.81).

3.1.2. Evaluation of Possible Anti-Hyperglycemic Activity In Vitro

The anti-hyperglycaemic activity of T. polium hydroalcoholic extract was evaluated
in vitro using the α-amylase inhibitory assay. The results illustrated in Figure 1 showed
that T. polium hydroalcoholic extract showed a pronounced anti-hyperglycemic activity at
the different assessed concentrations with IC50 of 111.68 µg/mL and thus exerted superior
activity compared to acarbose, the standard drug (IC50 = 3650.93 µg/mL).

Figure 1. α-Amylase inhibitory activity of various concentrations (µg/mL) of T. polium hydroal-

coholic extract (TPL) vs. acarbose; Values were expressed as means ± SD (n = 3) for three
parallel measurements.

3.1.3. Evaluation of Possible Anti-Alzheimer Activity In Vitro

Herein, T. polium hydroalcoholic extract was evaluated in vitro for its inhibitory
activity against AchE and BchE at different concentrations. Results illustrated in Figure 2
showed that T. polium showed excellent activity against BChE and AchE with IC50 values
of 28.69 and 4.93 µg/mL, respectively, showing values superior to that exhibited by
the standard galantamine with IC50 values of 34.75 and 6.27 µg/mL, for BchE and
AChE, respectively.
Life 2022, 12, 1579 9 of 23

Figure 2. Acetylcholinesterase (A) and butyrylcholinesterase (B) inhibitory activity of various concen-
trations (µg/mL) of T. polium hydroalcoholic extract (TPL) vs. Galantamine: Values were expressed
as means ± SD (n = 3) for three parallel measurements.

3.1.4. Evaluation of Possible Anti-Inflammatory Activity In Vitro

The anti-inflammatory activity of T. polium hydroalcoholic extract was evaluated
in vitro using a BSA denaturation assay. Table 2 shows that the plant extract exhibited a
strong anti-inflammatory effect with BSA denaturation inhibitory potential estimated by
97.53% at 2 mg/mL. This was closer to the inhibitory potential exerted by the standard
drug, diclofenac sodium, which showed 100% inhibition at 2 mg/mL.

Table 2. In vitro anti-inflammatory activity of various concentrations (µg/mL) of T. polium hydroal-

coholic extract (TPL) using BSA denaturation assay versus diclofenac sodium.

Concentration (µg/mL) TPL Diclofenac Sodium

2000 97.53 ± 0.47 100 ± 0.18
1000 95.76 ± 0.18 92 ± 0.15
500 91.03 ± 0.28 61 ± 0.15
250 80.35 ± 3.18 37 ± 0.18

3.1.5. Evaluation of the Anti-Inflammatory Activity In Vivo

Oral administration of Teucrium polium L. aerial parts hydroalcoholic extract to mice with
carrageenan-induced hind paw edema produced significant (p < 0.001) anti-inflammatory
Life 2022, 12, 1579 10 of 23

activity at the tested doses. The present study showed that the hydroalcoholic extract of
T. polium L. (10%) significantly reduced carrageenan-induced paw edema compared to the
reference drug, diclofenac sodium (10 mg/kg). A gradual decrease in edema paw weight
to 37.5% was observed compared to the standard drug diclofenac sodium, which exhibited
52.97% inhibition, as illustrated in Table 3. Thus, the obtained results confirmed the effect of
the extract as an anti-inflammatory agent that could be attributed to the synergistic action of
all of its biologically active phenolic compounds

Table 3. In vivo anti-inflammatory activity of T. polium hydroalcoholic extract (TPL) using

carrageenan-induced hind paw edema versus diclofenac sodium.

Average Paw Weight (g) % Oedema

Samples % Oedema
Left Right Reduction

Control
0.109 ± 0.006 0.076 ± 0.004 43.42% 0%
“Physiological water”
Standard
0.171 ± 0.009 0.142 ± 0.001 20.42% 52.97%
“Diclofenac sodium”
TPL 0.178 ± 0.001 0.140 ± 0.001 27.14% 37.49%

3.2. Functional Phenolic Substances Predominating in Teucrium polium L. Aerial Parts

The extract of T. polium L. aerial parts is highly rich in phenolic compounds. Previous
HPLC-UV-MS analysis conducted on the extract of Algerian Teucrium polium aerial parts
revealed the existence of many phenolic compounds belonging mainly to flavonoids
and caffeic acid derivatives. These include poliumoside (1), acteoside (2), hyperoside (3),
isoquercitrin (4), luteolin 7-O-β-D-glucopyranoside (5), diosmin (6), luteolin (7),
cirsiliol (8), cirsimaritin (9), cirsilineol (10), eupatorin (11), 5-desmethylsinensetin (12)
and salvigenin (13) [14]. A scheme showing compounds previously identified from
Algerian Teucrium polium aerial parts is illustrated in Figure 3.

3.3. Determination of the Total Content of Phenol and Flavonoids

The total content of phenol and flavonoids was determined using the Folin–Ciocalteu
method, where results revealed that Teucrium polium aerial parts were rich in polyphenols
with 36.35 ± 0.294 µg GAE/mg and in flavonoids with 24.30 ± 0.44 µg QE/mg. The
polyphenol content was expressed in mg of gallic acid, equivalent per gram of dry weight;
the flavonoid content was expressed in mg of quercetin equivalent per gram of dry weight.

3.4. Determination of Mineral Elements

The present study evaluated the mean values of elemental mineral concentrations in
Teucrium polium L. growing in Algeria. This was performed using the instrumental neutron
activation analysis method, and the results are demonstrated in Table 4. Nineteen minerals
comprising macro- and microelements, namely Ba, Br, Co, Cr, Cs, Eu, Fe, K, Mo, Na, Sb,
Sc, Sr, U and Zn, were determined by both techniques, which were INAA and ICP-OES.
The data presented in this experiment showed that six micronutrient elements “essential
chemical” were quantified in this plant analysis; their levels are arranged as follows;
K > Fe > Na > Zn > Cr > Co. However, two potential toxic elements were found, Br and Sb,
in addition to seven other elements.
Life 2022, 12, 1579 11 of 23

Figure 3. Scheme showing compounds previously identified from Algerian Teucrium polium aerial
parts [14].

Table 4. Results of chemical element’s mass fractions (mg/kg on dry mass basis) determined in
Teucrium polium L. by INAA and ICP-OES techniques.

Elements Teucrium polium L. The Used Technique

1 Ba 5.10 ± 0.67 INAA
2 Br 18.86 ± 11.56 INAA
3 Ca 18267 ± 386 ICP
4 Cl 219.5 ± 3 INAA
5 Co 0.480 ± 0.041 INAA
6 Cr 2.29 ± 0.65 INAA; ICP
7 Cs 0.111 ± 0.023 INAA
8 Eu 0.040 ± 0.015 INAA
9 Fe 983.9 ± 111.9 INAA; ICP
10 K 7619 ± 2295 INAA
11 Mg 230.2 ± 2 INAA; ICP
12 Mn 39.3 ± 5 INAA
13 Mo 0.28 ± 0.101 INAA
14 Na 803 ± 41 INAA
15 Sb 0.050 ± 0.013 INAA
16 Sc 0.012 ± 0.001 INAA
17 Sr 138.03 ± 7.97 INAA
18 U 0.040 ± 0.013 INAA
19 Zn 24.22 ± 1.04 INAA; ICP
Values were expressed as means ± SD (n = 3) for triplicate analyses, multiple irradiations or different photo peaks.
Life 2022, 12, 1579 12 of 23

3.5. In Silico Studies

3.5.1. Molecular Docking Validation Studies using Re-Docking and Superimposition
Validation experiments revealed an acceptable alignment between the docking pose of
the lead compounds that showed the best fitting with the co-crystallized lead conformers.
They displayed RMSD values of 1.18, 1.74, and 2.50 Å for human α-amylase, 5-lipoxygenase
and acetylcholine esterase, respectively (Figure S1).

3.5.2. Molecular Docking

Aiming to consolidate the results obtained from the in vitro studies and provide a
mechanistic postulation for the pronounced activity of Teucrium polium, molecular docking
was performed for previously identified compounds in the extract of Algerian T. polium
aerial parts on human α-amylase, 5-lipoxygenase and acetylcholine esterase (Table 5).

Table 5. Free binding energies (kcal/mol) of the main identified compounds in the Algerian T. polium
extract (TPL) used inside the active site of human α-amylase, 5-lipoxygenase and acetylcholine
esterase using in silico studies.

Compounds Human α-Amylase 5-Lipoxygenase Acetylcholine Esterase

Poliumoside (1) FD −10.10 FD
Acteoside (2) −35.50 −38.49 −18.48
Hyperoside (3) −30.53 −19.43 −17.22
Isoquercitrin (4) −33.79 −18.90 −13.99
Luteolin 7-O-β-D glucopyranoside (5) −29.49 −17.17 −24.29
Diosmin (6) 83.64 −9.54 −0.43
Luteolin (7) −47.89 −36.53 −49.55
Cirsiliol (8) −37.11 −40.04 −41.79
Cirsimaritin (9) −35.46 −15.93 −22.99
Cirsilineol (10) −35.50 −18.27 −25.67
Eupatorin (11) −25.95 −27.66 −34.66
5-Desmethylsinensetin (12) −23.38 −20.78 −30.77
Salvigenin (13) −23.56 −21.789 −30.08
Acarbose (Co-crystalized ligand) −75.26 ND ND
Nordihydroguaiaretic acid (NDGA)
ND −43.37 ND
(Co-crystalized ligand)
Donepezil (Co-crystalized ligand) ND ND −25.89
FD: fail to dock; ND: not done.

Regarding human α-amylase, luteolin (7) showed the best fitting, followed by
cirsiliol (8), displaying ∆G of −47.89 and −37.11 kcal/mol, respectively, approaching that
of acarbose, which showed ∆G of −75.26 kcal/mol. Luteolin forms one conventional H-
bond with Glu233; three π-anion bonds with Asp197, Asp300 and Tyr62; one π-π T-shaped
bond with His201; four π-alkyl bonds with Lys200, Leu162, Ile235 in addition to three salt
bridge interactions with His299, Arg195 and Lys200 (Figure 4A). However, cirsiliol forms
four conventional H-bonds with Gly306, His201 and Tyr151; one π-alkyl bond with Ile235
and one salt bridge interactions with Lys200 (Figure 4B). Acarbose (Co-crystalized ligand)
forms seven conventional H-bonds with His201, Tyr151, His305, Thr163, Gln63 and Trp59;
one alkyl interaction with Leu162 in addition to one C-H bond with Gly104 (Figure 4C).
Additionally, most of the examined compounds showed a promising degree of interaction
with the binding sites, such as acteoside (2), hyperoside (3), isoquercitrin (4), luteolin 7-O-β-D-
glucopyranoside (5), cirsimaritin (9), cirsilineol (10), eupatorin (11), 5-desmethylsinensetin (12)
and salvigenin (13), owing to the formation of multiple bonds with the amino acid existing at
the binding center such as H-bonds, π-π, π-alkyl and slat bridge interactions. The 2D binding
mode of major compounds identified from Algerian Teucrium polium aerial parts inside the
active site of human α-amylase was illustrated in Figure S2.
Life 2022, 12, 1579 13 of 23

Figure 4. The 2D and 3D binding modes of luteolin (A), cirsiliol (B) and acarbose (C) inside the active
site of human α-amylase; heavy green dotted bond, H-bonds; heavy pink dotted bond, π-π bonds;
light green dotted bond, C-H bonds; light pink dotted bond, π-alkyl bonds; dotted orange bonds, salt
bridge interactions.

Concerning 5-lipoxygenase, cirsiliol (8) followed by acteoside (2) revealed the most
firm fitting with the active sites displaying ∆G of −40.04 and −38.49 kcal/mol, respectively,
approaching that of nordihydroguaiaretic acid with ∆G of −43.37 kcal/mol. Cirsiliol forms
one H-bond with Asn554; one π–anion interaction with Glu172; one attractive charge
interaction with Lys173; three π–alkyl interactions with Leu607; three C-H bonds with
Asp176 and His367 (Figure 5A). However, acteoside forms two π–alkyl interactions with
Ala508 and Val504; two C-H bonds with Ala572; one π–anion interaction with Glu172
and four H-bonds with Gln963, Glu612 and His967 (Figure 5B). Nordihydroguaiaretic
acid (Co-crystalized ligand) forms two H-bonds with Ala606 and Gln609, one π–anion
interaction with Glu172, and two π–alkyl interactions with Leu607 (Figure 5C). In addition,
all other major tested compounds showed a pronounced fitting within the binding site that
is mainly attributed to the formation of plethora of bonds with the amino acid residues
Life 2022, 12, 1579 14 of 23

where the 2D binding mode of the major compounds identified from Algerian Teucrium
polium aerial parts inside the active site of 5-lipoxygenase was illustrated in Figure S3.

Figure 5. The 2D and 3D binding modes of cirsiliol (A), acetoside (B) and nordihydroguaiaretic acid (C)
inside the active site of 5-lipoxygenase, heavy green dotted bond, H-bonds; heavy pink dotted bond, π-π
bonds; light green dotted bond, C-H bonds; light pink dotted bond, π-alkyl bonds; dotted orange bonds,
π-anion interaction.

In addition, luteolin (7) showed the best fitting within acetylcholinesterase active
sites, followed by cirsiliol (8), displaying ∆G of −49.55 and −41.79 kcal/mol, respectively,
showing superior activity compared to donepezil, which showed ∆G of −25.89 kcal/mol.
Luteolin forms one H-bond with Phe295; one C-H bond with Ser203; one salt bridge with
His447; one π-anion with Trp286 in addition to six π–π interactions with Tyr341, Tyr124
and Phe338 (Figure 6A). Moreover, cirsiliol forms one salt bridge with His447; four π–π
interactions with Trp286, Tyr124 and Phe338; two H-bonds with Arg296, Phe225 and Ser203,
in addition to two C-H bonds with Ser293 and Tyr341(Figure 6B). However, donepezil,
the co-crystalized ligand, forms two π–cation interactions with Trp86 and Tyr337; three
π-π bonds with Tyr341, Trp286 and Trp86; one H-bond with Phe295; two C-H bonds with
Life 2022, 12, 1579 15 of 23

Ser293 and Tyr341 in addition to one π-δ bond with Tyr341 (Figure 6C). Thus, molecular
docking results further ascertained the in vitro results and highlighted that the secondary
metabolites of Teucrium polium target many crucial proteins. Consequently, Teucrium polium
showed pronounced biological activities. Concerning the rest of the major compounds,
they exert pronounced activity within the binding site, where the 2D binding mode of all
major compounds identified from Algerian Teucrium polium aerial parts inside the active
site of acetylcholine esterase is illustrated in Figure S4.

Figure 6. The 2D and 3D binding modes of luteolin (A), cirsiliol (B) and donepezil (C) inside the
active site of acetylcholine esterase; heavy green dotted bond, H-bonds; heavy pink dotted bond, π-π
bonds; light green dotted bond, C-H bonds; light pink dotted bond, π-alkyl bonds; purple dotted
bond, π-δ bond; dotted orange bonds, π-cation interaction.
Life 2022, 12, 1579 16 of 23

3.5.3. ADMET and Drug Likeness Prediction

ADMET (absorption, distribution, metabolism, excretion and toxicity) determination
for the prediction of the pharmacokinetic, pharmacodynamic and toxicity properties and
drug likeness was performed on previously identified phenolic compounds on Algerian
T. polium aerial parts extract. Results illustrated in Table 6 and Figure 7 revealed that
compounds (7–13) showed good human intestinal absorption consequently allocated in
the 99% absorption ellipse whereas compounds (1–6) showed very low human intestinal
absorption and concomitantly lie outside 99% absorption ellipse, as revealed in Figure 7.
Regarding solubility level, most of the tested compounds showed good and optimal
solubility except poliumoside (1), acteoside (2) and diosmin (6) that showed low to very
low but possible solubility. In addition, compounds (1–6) showed undefined, penetration
via BBB and hence placed outside the 99% BBB confidence eclipse in contrast to compounds
(7–13) that showed medium to low penetration via BBB lying inside 95 and 99% BBB
confidence eclipse. Additionally, compounds (1–6) showed less than 90% plasma protein
binding (PPB) in contrast to compounds (7–13) that revealed more than 90% PPB. In
addition, all of the examined compounds are non-CYP2D6 inhibitors. Unfortunately, most
of the compounds showed certain hepatic toxicity except compounds (1–4) that showed
no toxicity. Additionally, results illustrated in Table 7 reflected the suitability of most of
the examined compounds for their incorporation in different dosage forms, particularly
those that revealed promising biological activity as evidenced from their molecular weight
(M.W.), number of hydrogen bond acceptors (HBA), number of hydrogen bond donors
(HBD), number of rings, number of aromatic rings and number of rotatable bonds.

Table 6. ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of phenolic
compounds in Algerian T. polium aerial parts extract.

Absorption Solubility BBB PPB

Compounds CPY2D6 Hepatotoxic PSA-2D Alog p98
Level Level Level Level
Poliumoside (1) 3 1 4 False NI NT 308.782 −0.375
Acteoside (2) 3 2 4 False NI NT 249.29 0.484
Hyperoside (3) 3 3 4 False NI NT 203.585 −1.706
Isoquercitrin (4) 3 3 4 False NI NT 203.585 −1.706
Luteolin 7-O-β-D
3 4 4 False NI Toxic 182.77 −1.168
glucopyranoside (5)
Diosmin (6) 3 2 4 False NI Toxic 237.405 −0.395
Luteolin (7) 0 3 3 True NI Toxic 102.463 0.762
Cirsiliol (8) 0 3 3 True NI Toxic 103.022 1.674
Cirsimaritin (9) 0 3 3 True NI Toxic 85.722 2.619
Cirsilineol (10) 0 3 3 True NI Toxic 94.652 2.603
Eupatorin (11) 0 3 3 True NI Toxic 94.652 2.603
5-Desmethylsinensetin (12) 0 3 3 True NI Toxic 82.766 2.828
Salvigenin (13) 0 3 2 True NI Toxic 73.836 2.845
0, 1, 2, and 3 indicates good, moderate, low and very low absorption, respectively; 0, 1, 2, 3, 4, and 5 indicates
extremely low, very low but possible, low, good, optimal, and too soluble, respectively; 0, 1, 2, 3, and 4 denote
very high, high, medium, low, and undefined, penetration via BBB respectively. PBB, plasma protein binding;
false = less than 90%, True = more than 90%; NI: Non-inhibitor; NT: Non-toxic.
Life 2022, 12, 1579 17 of 23

Figure 7. ADMET Plot of Algerian T. polium aerial parts phenolic compounds displaying 95% and
99% confidence limit ellipses with respect to the human intestinal absorption and the blood–brain
barrier (BBB) models.

Table 7. Drug-likeness predictions of phenolic compounds in Algerian T. polium aerial parts extract
through using Biovia Discovery Studio software.

N◦ of Aromatic N◦ of Rotatable
Compounds ALogP M.W HBD HBA N◦ of Rings
Rings Bonds
Poliumoside (1) −0.375 770.728 11 19 2 5 13
Acteoside (2) 0.484 624.587 9 15 2 4 11
Hyperoside (3) −1.706 462.36 6 12 2 4 4
Isoquercitrin (4) −1.706 462.36 6 12 2 4 4
Luteolin 7-O-β-D
−1.168 446.361 5 11 2 4 4
glucopyranoside (5)
Diosmin (6) −0.395 608.545 8 15 2 5 7
Luteolin (7) 0.762 284.22 2 6 2 3 1
Cirsiliol (8) 1.674 329.281 2 7 2 3 3
Cirsimaritin (9) 2.619 314.289 2 6 2 3 3
Cirsilineol (10) 2.603 344.315 2 7 2 3 4
Eupatorin (11) 2.603 344.315 2 7 2 3 4
5-Desmethylsinensetin (12) 2.828 358.342 1 7 2 3 5
Salvigenin (13) 2.845 328.316 1 6 2 3 4

4. Discussion
Teucrium polium L. belonging to the family Lamiaceae is widely used in traditional
medicine to treat hypertension, diabetes, or as a wound-healing agent [13]. Results illus-
trated in this study showed that Teucrium polium aerial parts are rich in polyphenols with
36.35 ± 0.294 µg GAE/mg and in flavonoids with 24.30 ± 0.44 µg QE/mg. Variation in
geographical origin undoubtedly influences the total content of phenol and flavonoids in
the same species [35]. The extract of T. polium growing in Morocco demonstrated a higher
polyphenol content estimated by 95.53 ± 1.65 mg GAE/g in addition to a higher flavonoid
content reported to be 101.9 ± 1.97 mg RE/g (Rutin equivalent per gram of dry weight) [36].
Life 2022, 12, 1579 18 of 23

Furthermore, the extract of Tunisian T. polium revealed a lower flavonoid content that was
estimated to be 2.67 ± 0.05 mg RE/g [37]. In addition, T. polium L. aerial parts are highly
rich in phenolic compounds, as evidenced by HPLC-UV-MS analysis previously conducted
on the extract of Algerian Teucrium polium aerial parts that revealed the existence of many
phenolic compounds belonging mainly to flavonoids and caffeic acid derivatives [14].
Additionally, the evaluation of elemental mineral concentrations in Teucrium polium L.
growing in Algeria was conducted for the first time. Mineral elements, particularly the micro
and macro-nutrients are essential to various human metabolic processes and significantly
contribute to human health [38]. The essential elements of Na, Fe and K were detected as
the highest level among the other elements, where K showed the highest level, estimated at
7619 mg/kg, followed by Fe (984 mg/kg) and then Na (803 mg/kg). In addition, the concen-
tration of essential elements acting as micronutrients, such as Zinc, Chromium and Cobalt,
ranged from 24 to 0.48 mg/kg. Although two potential toxic elements were detected, their
levels lie below the tolerance limits compared with the recommended values (RDA) [39].
Moreover, Teucrium polium L. showed pronounced antioxidant, antihyperglycaemic,
anti-Alzheimer activity and anti-inflammatory activity exceeding the used standards. This
is mainly attributed to its richness with phenolic compounds, particularly flavonoids
highlighted by the detected total flavonoid contents and previously isolated compounds.
Flavonoids and phenolics are greatly popular as the largest phytochemical entities with
pronounced antioxidant properties from plants [40,41]. Regarding the antioxidant activity,
the obtained results follow what was previously reported by Sharififar et al. [42], which
reported the antioxidant potential of the Iranian T. polium hydroalcoholic extract owing
to its free radicle scavenging properties. However, herein, the reported results from the
Algerian species showed better antioxidant potential using DPPH assay from the previously
reported the Iranian species that showed the IC50 value of 20.1 ± 1.7 µg/mL. The variation
in the obtained results reflected the effect of geographical origin in altering to some extent
the biological activity that is, in turn, influenced by the secondary metabolites. Moreover,
flavonoids significantly prohibit oxidative stress-related diseases via scavenging of reactive
oxygen species (ROS) directly by different mechanisms comprising antioxidant enzymes
stimulation, inhibition of nitric oxide-induced oxidative stress in addition to metal-chelating
activity [43], whereas scavenging of free radicals is considered the most important mode
of antioxidant action of flavonoids as the polyphenol groups interrupt the free radical
chain reaction [44]. Regarding the structure–activity relationship, for efficient radical
scavenging behavior, the critical structural features are manifested by the presence of
an ortho-dihydroxy structure in ring B, which is critical for electron delocalization; the
existence of a 2,3 double bond in conjugation with a 4-keto function that allows the electron
delocalization from the B ring in addition to the presence of hydroxyl groups at positions
3 and 5 provides hydrogen bonding to the keto groups that are present in most of the
identified compounds in [44]. It is noteworthy to highlight that the antioxidant potential of
phenolic compounds and flavonoids is greatly influenced by functional group arrangement,
substitution, configuration, the number of hydroxyl groups that, in turn, affect metal ion
chelation ability and radical scavenging activity [45,46].
Furthermore, the pronounced antihyperglycaemic activity of Teucrium polium aerial
parts evidenced by an in vitro study and further supported by in silico studies greatly
relied upon the richness of Teucrium polium L. with flavonoids. It is worth highlighting
that T. polium hydroalcoholic extract showed one hundred and thirty times higher activity
than acarbose, which could be attributed to the richness of the plant with various phyto-
chemicals such as flavonoids, tannins, and saponins [47]. Previous studies on α-amylase
inhibitors identified from medicinal herbs revealed that several potent inhibitors belong
to the flavonoid class [32]. The obtained results further consolidated what was previously
reported by Dastjerdi et al. [48], showing that Iranian T. polium possesses a very good
activity in α-amylase inhibitory assay with IC50 values of 3.01 and 1.64 mg/mL for the
dichloromethane extract and with ethyl acetate extracts, respectively. Flavonoids elicited
an antihyperglycemic effect via suppressing α-glucosidase and α-amylase activities, at-
Life 2022, 12, 1579 19 of 23

tenuating insulin resistance and promoting pancreatic cell proliferation [5,49]. Moreover,
many studies further supported the results of in silico studies, where luteolin revealed
a potent antihyperglycemic effect via inhibition of α-amylase [50]. In addition, luteolin
showed an improved insulin action by direct activation of the PPAR pathway, by acting
at the insulin signaling pathway, and GLUT4 expression in addition to the up-regulation
of synaptic proteins expression and improving endothelial insulin resistance responsible
for inflammation [51]. Flavonoid antihyperglycemic activity may also elicit α-glucosidase
inhibition. This inhibition was greatly influenced by the hydroxylation and galloylation of
flavonoids that improved the inhibitory activity. On the contrary, the glycosylation of the
hydroxyl group and hydrogenation of the C2=C3 double bond on flavonoids weaken the
inhibition; however, caffeoylquinic acids showed strong prohibition of α-glucosidase [52].
Concerning the anti-Alzheimer activity of Teucrium polium aerial parts, it can be
concluded that T. polium could be used in the alleviation of Alzheimer’s evidenced by
its promising BChE and AChE inhibitory potential. This activity is perhaps due to its
richness in polyphenols since the members of Lamiaceae were found to be rich in phe-
nolic acids as active constituents that significantly contribute to their neuroprotective
properties [53]. Moreover, a study by Valdimir-Knežević et al. [54] on the ethanol extract
of several Lamiaceae cultivated in Croatia showed that T. polium was among the most
potent plant extracts, with significant AchE inhibitory rates exceeding 75% at 1 mg/mL
concentration. Alzheimer’s disease is a form of dementia that is characterized by the
presence of senile plaques, neurofibrillary tangles with concomitant synaptic loss and
neuronal death leading to gradual memory loss, a decrease in language skills in addition
to cognitive impairments [55]. The neurotransmitter acetylcholine has a crucial role in
learning process and memory in the hippocampus. Two enzymes, acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE), are involved in the of acetylcholine hydroly-
sis, decreasing its level in Alzheimer’s disease, and thus, prohibition of both enzymes
is a well-established strategy for the alleviation of Alzheimer’s disease [56]. Flavonoids
are promising natural products with neuroprotective potential that prevent or slow the
progression of Alzheimer disease via the inhibition of key enzymes such as AChE, BChE
and BACE-1 (Beta-site APP Cleaving Enzyme-1) [57]. Moreover, Shimmyo et al. showed
that flavonols and flavones are capable of inhibiting BACE-1 where the presence of OH
groups of C30 and C40 stabilized the binding poses of flavonoids within the BACE-1 active
center via hydrogen bond formation. Additionally, the existence of OH at C3 interacted in a
direct manner with the Asp catalytic residue, causing a notable enhancement of the BACE-1
inhibitory activity [58]. Moreover, previous studies reported the effectiveness of cirsiliol
as a neuroprotective agent and sedative acting as CNS depressants at the GABA chloride
channel and/or at glutamate binding sites that further supported the in silico studies [59].
Concerning the anti-inflammatory activity of Teucrium polium aerial parts, its pro-
nounced activity evidenced by BSA denaturation inhibitory potential estimated as 97.53%
at 2 mg/mL mainly relied upon its richness with flavonoids. It is worth highlighting
that water/ethanol is an effective solvent in extracting phytochemicals from the plant as
it combines polar and medium polarity properties [60]. Thus, it appears that the anti-
inflammatory effect of the extract may be due to the presence of flavonoids and phenolic
compounds in the plant [54,61]. Cirsiliol has been reported to possess a potent and selec-
tive 5-lipoxygenase inhibitory potential that further consolidated the in silico studies [62].
Several mechanisms of action have been suggested in an effort to interpret the mode of
action of flavonoids, such as their antioxidant potential, their ability to modulate the pro-
duction of proinflammatory cytokines and the expression of genes [63,64]. Thus, flavonoids
hinder the inflammation process by a reduction in cytokines and inflammatory markers
expression and via interaction with proteins incorporated in the incidence of inflammation.
Additionally, they modulate arachidonic acid (AA)-metabolizing enzymes activity, such as
COX, phospholipase A2 (PLA2), lipoxygenase (LOX), as well as the NO-producing enzyme
nitric oxide synthase (NOS) and thus reduce the production of AA, PG, leukotriene, and
NO, which are critical mediators of inflammation accounting for cellular mechanisms of
Life 2022, 12, 1579 20 of 23

anti-inflammation [65]. Luteolin was previously shown to inhibit the rat renal medulla COX
with an IC50 of 100–130 µM, whereas other flavonoids exhibited a significant inhibition
of LOX where the reduction in the C-2, 3-double bond and glycosylation reduced the
flavonoids inhibitory activities [63]. Thus, T. polium can act as a good candidate for alleviat-
ing many health-debilitating problems and can be highly beneficial in the pharmaceutical
industry and medical research.

5. Conclusions
Herein, Teucrium polium L. was evaluated for its antioxidant, antihyperglycaemic,
anti-Alzheimer activity and anti-inflammatory activity by different assays in vitro and
in vivo and then confirmed by in silico studies for the first time. In addition, the minerals
were investigated by two techniques, namely instrumental neutron activation analysis
(INAA) and inductively coupled plasma-optical emission spectrometry (ICP-OES), for
the first time as they are highly important, either micro or macronutrients, and essential
for various human metabolic processes. Results showed that Teucrium polium L. is rich in
minerals necessary for humans, such as calcium, potassium, iron, sodium, magnesium,
manganese and zinc. In addition, it showed promising antioxidant, antihyperglycaemic,
anti-Alzheimer activity and anti-inflammatory activity, which could be attributed to its
phenolic compounds as confirmed by in silico studies. Thus, it can be concluded that
T. polium can act as a good candidate for alleviating many health-debilitating problems
and could be incorporated into various pharmaceutical preparations. However, additional
in vivo and preclinical trials are highly recommended to ascertain the obtained results.
Additionally, performing docking experiments to examine the inhibitory potential of the
main compounds in Algerian T. polium extract versus butyrylcholinesterase and beta-
secretase 1 enzyme is highly recommended.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/life12101579/s1, Figure S1: Validation of the docking experiments
for human α-amylase, (A), 5-lipoxygenase (B) and acetylcholine esterase, (C); Figure S2: 2D binding
mode of compounds identified from Algerian Teucrium polium aerial parts inside the active site of
human α-amylase; Figure S3: 2D binding mode of compounds identified from Algerian Teucrium
polium aerial parts inside the active site of 5-lipoxygenase; Figure S4: 2D binding mode of compounds
identified from Algerian Teucrium polium aerial parts inside the active site of acetylcholine esterase.
Author Contributions: Conceptualization, N.B. and M.M.; methodology, N.B. and M.M.; software,
F.S.Y.; validation, N.B., M.M. and M.B.; formal analysis, I.L. and H.O.; investigation, I.L. and H.O.;
resources D.M.A. and R.H.H.; data curation, N.B., M.M., A.R. and M.A.F.; writing—original draft
preparation, A.B., S.B. (Samir Bagaa), S.B. (Siham Boubekeur) and A.B.; writing—review and editing,
F.S.Y.; supervision, N.B. and F.S.Y.; funding acquisition, D.M.A. and R.H.H. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz
University (KAU), Jeddah, Saudi Arabia, under grant number (RG-23-166-43).
Institutional Review Board Statement: The research protocol (use of experimental animals) used
was based on the Declaration of Helsinki, The Council for International Organizations of Medical
Sciences (CIOMS), and these protocols have been approved for the application of ethical health
research with the Algerian Executive Directive (18 March 2004, N◦ 10–90 JORA) and accordance also
to the Law No.88-08 of 26 January 1988 relating to veterinary medicine activities and the protection
of animal health (N◦ JORA: 004 of 27-01-1988).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available in the manuscript.
Acknowledgments: The Scientific Research (SR) at King Abdulaziz University (KAU), Jeddah,
Saudi Arabia, has funded this project under grant no. (RG-23-166-43). Therefore, all the authors
acknowledge, with thanks, the SR for technical and financial support.
Conflicts of Interest: The authors declare no conflict of interest.
Life 2022, 12, 1579 21 of 23

Abbreviations

ACHE acetylcholinesterase
BChE butyrylcholinesterase
DPPH 2,2-diphenyl-1-picrylhydrazyl
GOR galvinoxyl radical
INAA instrumental neutron activation analysis
ICP-OES inductively coupled plasma—optical emission spectrometry

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