Molecules 29 00849 v2

molecules
Article
Chemical Composition Antioxidant and Anti-Inflammatory
Activities of Myrtus communis L. Leaf Extract: Forecasting
ADMET Profiling and Anti-Inflammatory Targets Using
Molecular Docking Tools
Samia Belahcene 1 , Widad Kebsa 2 , Tomilola Victor Akingbade 3 , Haruna Isiyaku Umar 3 ,
Damilola Alex Omoboyowa 4 , Abdulaziz A. Alshihri 5 , Adel Abo Mansour 6 , Abdulaziz Hassan Alhasaniah 7 ,
Mohammed A. Oraig 8 , Youssef Bakkour 5, * and Essaid Leghouchi 1, *
1 Laboratory of Biotechnology, Environment and Health, Faculty of Nature and Life Sciences, University of Jijel,
Jijel 18000, Algeria
2 Laboratory of Molecular Toxicology, Faculty of Nature and Life Sciences, University of Jijel, Jijel 18000, Algeria;
w.kebsa@univ-jijel.dz
3 Computer-Aided Therapeutic Discovery and Design Platform, Federal University of Technology,
PMB 704 Akure, Gaga 340110, Nigeria; victomilola@gmail.com (T.V.A.); uhumar@futa.edu.ng (H.I.U.)
4 Phyto-Medicine and Computational Biology Laboratory, Department of Biochemistry, Adekunle Ajasin
University, Akungba Akoko 57257, Nigeria
5 Department of Radiological Sciences, College of Applied Medical Science, King Khalid University,
Abha 61421, Saudi Arabia; aaalshehre@kku.edu.sa
6 Department of Clinical Laboratory Sciences, College of Applied Sciences, King Khalid University,
Abha 61421, Saudi Arabia
7 Department of Clinical Laboratory Sciences, College of Applied Sciences, Najran University,
Citation: Belahcene, S.; Kebsa, W.; Najran 1988, Saudi Arabia
8 Radiology Department, Khamis Mushayt General Hospital, Khamis Mushayt 62433, Saudi Arabia;
Akingbade, T.V.; Umar, H.I.;
moraig@moh.gov.sa
Omoboyowa, D.A.; Alshihri, A.A.;
* Correspondence: ybakkour@kku.edu.sa (Y.B.); leghouchi_e@univ-jijel.dz (E.L.)
Abo Mansour, A.; Alhasaniah, A.H.;
Oraig, M.A.; Bakkour, Y.; et al.
Chemical Composition Antioxidant
Abstract: Compounds derived from natural sources continue to serve as chemical scaffolds for
and Anti-Inflammatory Activities of designing prophylactic/therapeutic options for human healthcare. In this study, we aimed to
Myrtus communis L. Leaf Extract: systematically unravel the chemical profile and antioxidant and anti-inflammatory activities of
Forecasting ADMET Profiling and myrtle methanolic extract (MMEx) using in vitro, in vivo, and in silico approaches. High levels
Anti-Inflammatory Targets Using of TPC (415.85 ± 15.52 mg GAE/g) and TFC (285.80 ± 1.64 mg QE/g) were observed. Mass
Molecular Docking Tools. Molecules spectrophotometry (GC-MS) analysis revealed the presence of 1,8-cineole (33.80%), α-pinene (10.06%),
2024, 29, 849. https://doi.org/ linalool (4.83%), p-dimethylaminobenzophenone (4.21%), thunbergol (4%), terpineol (3.60%), cis-
10.3390/molecules29040849 geranyl acetate (3.25%), and totarol (3.30%) as major compounds. MMEx induced pronounced
Academic Editor: Antonio dose-dependent inhibition in all assays, and the best antioxidant activity was found with H2 O2 , with
Palumbo Piccionello an IC50 of 17.81 ± 3.67 µg.mL−1 . MMEx showed a good anti-inflammatory effect in vivo by limiting
the development of carrageenan-induced paw edema. The pharmacokinetic profiles of the active
Received: 25 November 2023
molecules were determined using the SwissADME website, followed by virtual screening against
Revised: 29 January 2024
anti-inflammatory targets including phospholipase A2 (PLA-2), cyclooxygenase-2 (COX-2), tumor
Accepted: 30 January 2024
Published: 14 February 2024
necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and NF-κB. A pharmacokinetic study revealed
that the molecules have good absorption, distribution, and metabolism profiles, with negative organ
toxicity. Among the compounds identified by GC-MS analysis, pinostrobin chalcone, cinnamyl
cinnamate, hedycaryol, totarol, and p-dimethylaminobenzophenone were observed to have good
Copyright: © 2024 by the authors. binding scores, thus appreciable anti-inflammatory potential. Our study reveals that MMEx from
Licensee MDPI, Basel, Switzerland. Algerian Myrtus communis L. can be considered to be a promising candidate for alleviating many
This article is an open access article
health complaints associated with oxidative stress and inflammation.
distributed under the terms and
conditions of the Creative Commons
Keywords: Myrtus communis L.; methanolic extract; bioactive molecules; antioxidant; anti-inflammatory;
Attribution (CC BY) license (https://
molecular docking; ADMET
creativecommons.org/licenses/by/
4.0/).
Molecules 2024, 29, 849. https://doi.org/10.3390/molecules29040849 https://www.mdpi.com/journal/molecules

Molecules 2024, 29, 849 2 of 32
1. Introduction
Even though free radicals can operate as redox-signaling messengers, oxidative stress
can also orchestrate the complex pathophysiological mechanisms in many diseases [1]. An
imbalance between pro-oxidant and antioxidant molecules affects the redox circuitry and
cell integrity through a toxic onslaught on non-target tissues [2]. A stochastic accumulation
of reactive oxygen species (ROS) and their precursors stimulates the expression of redox-
sensitive pro-inflammatory cytokines and caspases [3]. Oxidative stress and inflammation
are interrelated in an ambivalent way, since one can promote the other, leading to a “toxic
feedback” system [4]. An acute inflammatory cascade is a protective process characterized
by a spatiotemporal orchestration of enzymatic reactions and cellular activation [5]. It
starts abruptly and generally resolves quickly [6], resulting in tissues being returned to
functional homeostasis with an endogenous programmed resolution [7,8]. It progresses
through the generation of a large pool of arachidonic acid (AA) from membrane phospho-
lipids by the action of phospholipase A2 (PLA2) [9]. Subsequently, AA is metabolized by
cyclooxygenase (COX), resulting in the production of eicosanoids such as leukotrienes
(LTs), prostaglandins (PGs), and thromboxane (TX) [10]. The primary inflammatory stim-
uli, including interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α), which are
cell-signaling proteins (cytokines) responsible for the acute phase reaction by activating
typical NF-κB signaling and related receptors, are closely linked to oxidative stress mecha-
nisms [11]. If acute inflammation is not resolved, it becomes destructive, more complex,
and sophisticated [12]. This scenario is likely to amplify drastically due to the prevalence of
uncontrollable increased cytokine release (cytokine storm), leading to long-lasting inflam-
mation [13]. Chronic inflammation also produces free radicals, which ultimately aggravate
the inflammatory status [14].
In the pharmaceutical industry, archetypal non-steroidal anti-inflammatory drugs
(NSAIDs) have been extremely popular for their certified ability to prevent inflammation;
they do this by extinguishing COXs, which in turn inhibit the formation of PGs and PLA2
to suppress the production of other pro-inflammatory mediators [15,16]. The unrestrained
increase in the use of NSAIDs over the years is still controversial, since there are an escalat-
ing incidences of toxicity [17]. Unexpectedly, adverse gastrointestinal reactions, including
occult bleeding, so-called enteropathy [18], kidney failure [19], and liver damage [20], were
reported to be the most pervasive and unavoidable side effects of the prolonged consump-
tion of NSAIDs, putting people all over the world in jeopardy. Notably, the alarming
spread of adverse toxicity in certain segments of the population, the high cost, and the
upsurge in drug resistance are three specific hindrances to increasing the use of NSAIDs,
which are becoming less supportive with a narrow safety window. With an increased
collective consciousness among people, a belief has taken root that everything “natural”
is undoubtedly healthy [21]. Against the backdrop of these contrasting circumstances, a
number of research teams are eagerly searching for a prophylactic therapeutic strategy
from nature that has a significant safety margin when applied to both experimental animals
and humans [22,23].
Myrtus communis L. (family: Myrtaceae) is a useful ethnomedicinal shrub explicitly
grown in districts in Algeria that are native to Mediterranean ecosystems, with a broad
ecological amplitude adapted to various climatic conditions [24]. Historically, it was widely
used as medicine under the vernacular name “El-Rayhan”. Areal parts of myrtle have been
used as a decoction, an infusion, and a health remedy for bathing newborns with inflamed
skin and washing sores [25,26]. Additionally, it was used to treat oral wounds, disorders
of the digestive and urinary systems [25], diarrhea, peptic ulcers, hemorrhoids [27], and
inflammations [28,29]. Due to its qualified safety, there have been intensified efforts to
determine the chemical profile and various biological effects of myrtle extracts. Hence, the
ethnopharmacological value of this plant derives from its ubiquitously abundant phyto-
chemicals, including poly(phenols), flavonoids, tannins, gallic acid, flavonol derivatives,
hydroxybenzoic acids alkaloids, terpenoids, and quinonoids [30]. Under rigorous scientific
scrutiny, promising antibacterial [31], anti-inflammatory [28,32], anticancer [33], antioxi-
Molecules 2024, 29, 849 3 of 32
dant, antidiabetic [24], antigenotoxic [34], and antimicrobial activities [35] of M. communis
were discovered with multi-component, multi-target, and multi-mechanism models, mak-
ing it an ideal choice to be exploited for the development of novel therapies [36]. Myrtle has
been deeply scrutinized by the research community, whereas in Algeria, the chemical pro-
file and biological activities of myrtle leaves have not been explicitly investigated. There is
no plausible evidence explaining its anti-inflammatory behavior at the molecular level. This
study aims to elucidate these mechanisms in order to bridge the research gap and provide
deep insights into how MMEx can be harnessed for specific therapeutic applications.
The exceptional advancements in computational approaches represent an opportunity
to identify and design pharmacologically active natural molecules that can target proteins
of interest [37]. The use of in silico tools has recently opened new avenues for research in
the realm of pharmacotoxicology. Consequently, there is currently an increased focus on
developing effective, safe, and low-cost therapeutic modalities with the help of chemin-
formatics tools, including pharmacokinetic and molecular docking, in order to provide
valuable insights into complex and experimentally difficult phenomena such as enzyme
reaction mechanics and ligand–receptor linkages [6,38].
The objective of this study is to provide a holistic and comprehensive view of the
pharmacological potential of MMEx, first by deciphering and identifying the phytochemical
composition, and secondly by evaluating the in vitro, in vivo, and in silico antioxidant
and anti-inflammatory activities involved. Furthermore, the ADMET profiling and drug-
likeness of the identified phytochemicals are rationalized using molecular docking tools.
2. Results
2.1. Extraction Yeild (EY) and Total Polyphenol, Flavonoid, and Tannin Contents
The extraction yield of Myrtus communis L. methanolic extract is 34.30%. Table 1 shows
the contents of polyphenols, flavonoids, and tannins in the methanolic extract of Myrtus
communis L. The data clearly show considerable amounts of all three.
Table 1. Contents of total polyphenols, flavonoids, and tannins in Myrtus communis L. methanolic extract.
Polyphenols Flavonoids Condensed Hydrolysable

Sample
(mg EAG.g−1 ) (mg QUE. g−1 ) Tannins (CT) (%) Tannins (HT) (%)
MMEx 415.85 ± 15.52 285.80 ± 1.64 0.90 0.68
2.2. GC-MS Analysis

GC-MS is a key technological platform that is well established for providing a some-
what clearer picture of pharmaceutical ingredients [39]. The chemical profile of the in-
vestigated plant extract, which is referred to as the “fingerprint” of traditional Algerian
medicine, can be clearly reflected by a chromatogram.
The results of the GC-MS analysis of MMEx are presented as a chromatogram in
Figure 1. The compounds are listed according to their mass/charge ratio (m/z) in Table 2.
A total of 57 bioactive compounds were putatively identified. The predominant compound
in myrtle extract was 1,8 cineol (33.80%). Alpha-pinene constituted 10.06%, followed by
linalool (4.83%) and elaidic acid, and isopropyl ester (4.21%). The extract of myrtle con-
tains flavonoids, including pinostrobin chalcone, and phenolic acid derivatives, including
cinnamyl cinnamate.
2.3. Antioxidant Activity

The ability to remove free radicals by MMEx is dependent on the polyphenolic con-
tent. It was clearly observed that the presence of a high phenolic and flavonoid content
was in harmony with the antioxidant activity exerted by MMEx in all antioxidant tests.
However, in high concentrations (100–200 µg.mL−1 ), there was no significant change in
inhibition, probably because the critical concentration of phenols is sufficient to achieve
Molecules 2024, 29, 849 4 of 32
Molecules 2024, 29, x FOR PEER REVIEW 4 of 33

the desired quenching power, and the over-saturation concentration has no effect on the
antioxidant level.
Figure 1. Representative GC-MS chromatogram of Myrtus communis L. methanolic extract. Peak

Figure 1. Representative GC-MS chromatogram of Myrtus communis L. methanolic extract. Peak
numbers correspond to compound numbers in Table 2.
numbers correspond to compound numbers in Table 2.
Table 2. Chemical composition of MMEx by GC–MS. Rt, retention time.
Table 2. Chemical composition of MMEx by GC–MS. Rt, retention time.
Chemical
Peak Compound Chemical (%) Rt m/z
Peak Compound Formula (%) Rt m/z
Formula
1 Alpha-pinene C10H16 10.06 3.079 93.05
1 Alpha-pinene C10 H16 10.06 3.079 93.05
22 Isobutyl isobutyrate
Isobutyl isobutyrate C8 H16 O2 C8H16O0.232 71.05
0.233.477 3.477 71.05
33 D-limonene
D-limonene C10 H16 C10 H 16 0.52 68.05
0.525.365 5.365 68.05
44 Iso-butyl-2-methylbutyrate
Iso-butyl-2-methylbutyrate C9 H18 O2 C9H18O0.232 57.05
0.235.529 5.529 57.05
55 1,8-Cineole
1,8-Cineole C10 H18 O C10H18O 33.8 33.85.883 5.883 43.00
43.00
6 2-Methylbutyl 2-methylbutyrate C10 H20 O2 0.26 7.920 85.05
6 2-Methylbutyl 2-methylbutyrate C10H20O2 0.26 7.920 85.05
7 Linalool C10 H18 O 4.83 8.687 71.05
78 Linalool
Trans-pinocarveol C10 H16 O C10H18O 0.18 71.05
4.839.585 8.687 92.05
89 Trans-pinocarveol
Phenylethyl alcohol C8 H10 O C10H16O 0.31 92.05
0.1810.073 9.585 71.05
910 (-)-Terpinen-4-ol
Phenylethyl alcohol C H
10 18 O C8H10O0.16 0.3110.310 91.05
10.073 71.05
Estragole C10 H12 O 0.19
11
10 (-)-Terpinen-4-ol C10H18O 0.1610.465 10.310148.15
91.05
12 Terpneol C10 H18 O 3.60 10.724 59.05
11
13 Estragole
Linalyl acetate C12 H20 O2 C10H12O 0.85 148.15
0.1911.234 10.46593.10
12
14 Terpneol
(-)-Cis-carveol C10 H16 O C10H18O 0.13 59.05
3.6011.543 10.724109.10
15
13 (+-)-Pulegone
Linalyl acetate C10 H16 O C12H20O 0.16
2 0.8511.663 11.23481.05
93.10
16
14 Geraniol
(-)-Cis-carveol C10 H18 O C10H16O 1.00 0.1312.053 69.10
11.543 109.10
17 Terpinyl acetate C12 H20 O2 0.64 12.976 121.10
15
18 (+-)-Pulegone
Geranyl acetate C12 H20 O2 C10H16O 0.16 81.05
0.1613.247 11.66343.00
16
19 Geraniol
Caryophyllene C15 H24 C10H18O 0.49 69.10
1.0013.505 12.05393.10
1720 Terpinyl acetate
Ethanone, 1-(2-hydroxy-5-methylphenyl)- C12 H20 O2 C12H20O 3.25
2 0.6413.595 12.976 121.10
69.10
1821 Coumaran
Geranyl acetate C H
9 10 2O C12H20O 0.14
2 0.1613.848 13.247150.10
43.00
22 Alpha-caryophyllene C8 H8 O 0.19 13.970 120.05
19 Caryophyllene C15H24 0.49 13.505 93.10
23 Chavibetol C15 H24 0.25 14.144 93.10
2024 Ethanone,
Methyl1-(2-hydroxy-5-methylphenyl)-
eugenol C10 H12 O2 C12H20O 2
2.48 69.10
3.2514.258 13.595164.10
2125 Coumaran
(Hydroxymethyl)ethylene acetate C11 H14 O2 C9H10O 1.25
2 150.10
0.1414.649 13.848178.10
2226 Cyclohexanecarboxaldehyde,
Alpha-caryophyllene C7 H12 O2 C8H8O0.17 0.1915.334 43.00
13.970 120.05
6-methyl-3-(1-methylethyl)-2-xo-1-(3-oxobutyl)- C15 H24 O3
23 Chavibetol C15H240.19 0.2515.985 14.144139.10
93.10
27 2,4-Hexanedione, 5-methyl-3-(2-methyl-1-ropenyl)- C11 H18 O2 0.12 16.626 43.05
2428 Methyl eugenol
Durohydroquinone C10 H14 O2 C10H12O 2
0.67 164.10
2.4816.971 14.258166.10
25 (Hydroxymethyl)ethylene acetate C11H14O2 1.25 14.649 178.10
26 Cyclohexanecarboxaldehyde, 6-methyl-3-(1-methylethyl)-2-xo-1-(3-oxo-C7H12O2 0.17 15.334 43.00
butyl)- C15H24O3 0.19 15.985 139.10
27 2,4-Hexanedione, 5-methyl-3-(2-methyl-1-ropenyl)- C11H18O2 0.12 16.626 43.05
Molecules 2024, 29, 849 5 of 32
Table 2. Cont.
Chemical
Peak Compound (%) Rt m/z
Formula
29 O-eugenol C10 H12 O2 0.55 17.100 164.10
30 Caryophyllene oxide C15 H24 O 0.14 17.222 43.00
31 Cedrol C15 H26 O 0.21 17.370 95.10
32 Hedycaryol C15 H26 O 0.41 18.225 59.05
33 1, 2,4-Cyclopentanetrione, 3-(2-pentenyl)- C10 H12 O3 0.30 18.475 180.10
34 Benzaldehyde, 2-hydroxy-4-methyl- C8 H8 O2 0.27 18.697 136.05
35 2-Pentadecanone, 6, 10,14-trimethyl-(or phytone) C18 H36 O 0.45 20.321 43.00
36 2-Naphthalenecarboxylic acid, 3,4-dihydro- C18 H36 O 0.25 21.152 129.10
37 β-selinenol C15 H26 O 0.36 21.394 149.10
38 2-O-tosyl-1,3,4,6-tetra-o-acetyl-alpha-d-galactose C21 H26 O12 S 0.28 21.492 91.10
39 Isobutyl phthalate C16 H22 O4 0.34 21.590 149.05
40 Isopropyl palmitate C19 H38 O2 2.44 22.032 43.05
41 Palmitic acid C16 H32 O2 2.97 22.656 73.05
42 Dibutyl phthalate C16 H22 O4 0.17 22.793 149.05
43 1-Naphthalene propanol alp C20 H34 O 0.73 23.182 81.05
44 Cadalene C15 H18 0.16 23.305 183.10
45 p-Dimethylaminobenzophenone C15 H15 NO 4.21 23.680 326.15
46 Elaidic acid, isopropyl ester C21 H40 O2 2.01 24.082 55.00
47 Isopropyl stearate C21 H42 O2 0.19 24.256 102.05
49 Tetracosane C24 H50 0.58 24.321 57.10
50 Oleic acid C20 H42 1.78 24.714 55.05
51 Sulfuric acid, octadecyl 2-propyl ester C21 H44 O3 S 1.26 25.355 57.05
52 Eicosane C18 H34 O2 1.70 26.500 57.05
53 Thunbergol C20 H34 O 4.00 26.647 81.10
54 Totarol C20 H30 O 3.30 26.850 271.20
55 Cinnamyl cinnamate C18 H16 O2 0.23 28.726 131.05
56 Pinostrobin chalcone C16 H14 O4 1.80 29.347 270.10
57 Tetratriacontane C34 H70 2.42 29.597 57.10
Total - 99.72 - -
During the experiment, a change in DPPH intensity was visually apparent, as the color
changed from deep violet to yellow. Inhibition increased from 8.97 ± 1.63% at 12.5 µg.mL−1
to 88.25 ± 0.40% at 200 µg.mL−1 (Figure 2A), with an IC50 of 33.08 ± 1.86 µg.mL−1 , which
was comparable to ascorbic acid (IC50 of 35.02 ± 2.57 µg.mL−1 ) with no significant difference.
Furthermore, MMEx exhibited a strong H2 O2 radical scavenging ability compared to
the reference molecule, with inhibition increasing from 54.89 ± 1.28% to 62.70 ± 1.22% and
from 54.90 ± 2.79% to 84.16 ± 2.73% for MMEX and ascorbic acid, respectively, at 12.5 to
200 (µg.mL−1 ) concentration (Figure 2B). The IC50 values of MMEx and ascorbic acid were
17.81 ± 3.69 and 15.23 ± 1.01 µg.mL−1 , respectively.
As shown in Figure 3A, the hydroxyl radical OH-neutralizing test showed that
MMEx had significantly higher (p < 0.05) antioxidant activity, with an IC50 value of
11.52 ± 0.20 µg.mL−1 , which is 2.65 times higher compared to the reference molecule
(30.50 ± 1.29 µg mL−1 ).
The blue-green ABTS•+ solution turned pale yellow and then colorless. MMEx
reduced the ABTS•+ species in a dose-dependent manner, with inhibition increasing
from 56 ± 3.18% at 12.5 µg.mL−1 to 85.27 ± 0.87 at 200 µg.mL−1 (Figure 3B). This excellent
power in capturing ABTS•+ species was reflected by an IC50 of 22.83 ± 0.56 µg.mL−1 ,
which is statistically significant (p < 0.05) and 3.9 times higher compared to the reference
standard ascorbic acid (87.62 ± 0.50 µg mL−1 ).
Molecules 2024, 29, 849 6 of 32
Figure 2. Antioxidant properties of MMEx and ascorbic acid (reference) by (A) DPPH and (B) H2 O2 ,
and their IC50 values (b, d, f, h, and j, respectively). Data are expressed as mean ± SEM (n = 3). Ns,
no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001. (t test) using GraphPad Prism 8 for
Microsoft Office 10.
Figure 3C illustrates the capacity to transform a ferric ion (Fe3+ ) into a ferrous ion
(Fe2+ ),
creating a chromogenic complex. The samples underwent a change in color from
yellow to pale/light green or Prussian blue in the solution, and a high level of FRAP-
capturing activity by MMEx was noted, as expected. MMEx displayed 77.91 ± 0.54%
inhibition against ascorbic acid and 85.09 ± 0.34% at the highest tested concentration
(200 µg.mL−1 ). The IC50 value of MMEx and the standard was 47.09 ± 4.41 µg and
28.80 ± 0.74 µg.mL−1 , respectively.
2.4. Anti-Inflammatory Activity

2.4.1. In Vitro: Inhibition of BSA Denaturation
As a part of the inquiry into the mechanism of anti-inflammatory action, the potential
of MMEx to suppress the denaturation of proteins was explored. The inhibitory effects of
various concentrations of MMEx with the highest polyphenol content on protein denatura-
tion are depicted in Figure 4. A concentration-dependent inhibition of BSA denaturation
via MMEx in the range of 12.5–200 µg.mL−1 was noted. MMEx at 200 mg.kg−1 achieved a
highly significant (p < 0.001) inhibition of BSA denaturation of (58.27 ± 0.19%) in a dose-
dependent manner. The anti-inflammatory activity of MMEx in vitro was comparable to
Molecules 2024, 29, 849 7 of 32
that of the well-known nonsteroidal anti-inflammatory drug diclofenac (DCF). A significant

difference
Molecules 2024, 29, x FOR PEER REVIEW in the inhibition of thermally induced protein denaturation was observed with
7 of 33
MMEx, reflected by an IC50 value of 23.86 ± 0.31 µg.mL−1 , which is 2.43 times lower than
the IC50 of DCF (9.82 ± 3.14 µg.mL−1 ).
Figure
Figure3.3.
Antioxidant properties
Antioxidant of MMEx
properties of MMExandand
ascorbic acidacid
ascorbic (reference) by (A)
(reference) byhydroxyl radical,
(A) hydroxyl (B)
radical,
ABTS, and (C) FRAP and their IC 50 values. Data are expressed as mean ± SEM (n = 3). ns, no signif-
(B) ABTS, and (C) FRAP and their IC50 values. Data are expressed as mean ± SEM (n = 3). ns,
icant difference. * p < 0.05, ** p < 0.01, *** p < 0.001 (t-test) using GraphPad Prism 8 for Microsoft
no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001 (t-test) using GraphPad Prism 8 for
Office.
Microsoft Office.
The blue-green ABTS•+ solution turned pale yellow and then colorless. MMEx re-
duced the ABTS•+ species in a dose-dependent manner, with inhibition increasing from
56 ± 3.18% at 12.5 µg.mL−1 to 85.27 ± 0.87 at 200 µg.mL−1 (Figure 3B). This excellent power
in capturing ABTS•+ species was reflected by an IC50 of 22.83 ± 0.56 µg.mL−1, which is
via MMEx in the range of 12.5–200 µg. mL−1 was noted. MMEx at 200 mg.kg−1 achieved a
highly significant (p < 0.001) inhibition of BSA denaturation of (58.27 ± 0.19%) in a dose-
dependent manner. The anti-inflammatory activity of MMEx in vitro was comparable to
that of the well-known nonsteroidal anti-inflammatory drug diclofenac (DCF). A signifi-
cant difference in the inhibition of thermally induced protein denaturation was observed
Molecules 2024, 29, 849 with MMEx, reflected by an IC50 value of 23.86 ± 0.31 µg.mL−1, which is 2.43 times lower 8 of 32
than the IC50 of DCF (9.82 ± 3.14 µg.mL−1).
Figure Figure 4. Inhibition

4. Inhibition of heat-inducedBSA
of heat-induced BSA denaturation.
denaturation. Data are expressed
Data as mean
are expressed SEM (n±= SEM
as±mean 3). (n = 3).
DCF was used as positive control. Mean values of samples showing significant differences com-
DCF was used as positive control. Mean values of samples showing significant differences compared
pared to control (untreated 5% BSA water solution). *** p < 0.001 (t-test) using GraphPad Prism 8 for
to control (untreated
Microsoft Office. IC5% BSA water
50 analysis solution).
performed *** p < Prism
using GraphPad 0.0018.(t-test) using GraphPad Prism 8 for
Microsoft Office. IC50 analysis performed using GraphPad Prism 8.
2.4.2. In Vivo: Carrageenan-Induced Paw Edema in Rats
2.4.2. In Vivo:
Paw Carrageenan-Induced
edema in the experimentalPaw Edema
groups in Rats
is shown in Figure 5. A significant increase
in paw
Paw volume
edema in was
the observed in all groups
experimental groupsthatisreceived
shownsub-plantar
in Figureinjections of 1% Car increase
5. A significant
solution compared to the control group 2 h after injection, and the increase was sustained
in paw volume was observed in all groups that received sub-plantar injections of 1%
throughout the experiment. The increase in paw volume was lower in rats that received
Car solution
DCF andcompared to the control
the investigational polyphenolicgroup 2 h after
fraction of MMExinjection,
at bothand
doses the(25increase
and33 was
Molecules 2024, 29, x FOR PEER REVIEW
sustained throughout the experiment. The increase in paw volume was lower9 of in rats that
received DCF and the investigational polyphenolic fraction of MMEx at both doses (25
and 50 mg.kg−−11 ) compared to the untreated positive control (Car). The inhibitory effect of
50 mg.kg ) compared to the untreated positive control (Car). The inhibitory effect of the
the lower dose
lower dose(25
(25mg/kg)
mg/kg) ofof MMEx
MMEx lastedlasted for a shorter
for a shorter time
time than than of
the effect thetheeffect
higherofdose
the higher
dose (50(50 mg/kg). At the end of the experiment, the inhibitory effect of the highest dose of MMEx dose of
mg/kg). At the end of the experiment, the inhibitory effect of the highest
MMEx(50 (50mg.kg
mg.kg −1 ) did not differ significantly from the effect of DCF. The lower dose of
−1) did not differ significantly from the effect of DCF. The lower dose of MMEx
(25 mg/kg) inhibited
MMEx (25 mg/kg) inhibited paw edema
paw to a lesserto
edema extent than extent
a lesser DCF andthan
the higher
DCF dose
and oftheMMEx.
higher dose
The reference drug, DCF, showed a biphasic pattern and its highest inhibition
of MMEx. The reference drug, DCF, showed a biphasic pattern and its highest inhibition (69.83 ±
6.74%; mean ± SEM) occurred at 4 h (p < 0.01). MMEx showed a similar inhibition of paw
(69.83 ± 6.74%; mean ± SEM) occurred at 4 h (p < 0.01). MMEx showed a similar inhibition
edema, with the highest inhibition recorded as 58.26 ± 3.41% at the forth h (p < 0.01).
of paw edema, with the highest inhibition recorded as 58.26 ± 3.41% at the forth h (p < 0.01).
5. (A)5.Influence
Figure Figure of MMEx
(A) Influence of MMEx(25
(25and
and 50
50 mg/kg)
mg/kg) onon Car-induced
Car-induced paw paw
edema.edema. Data represent
Data represent
percentage
percentage of increased
of increased pawpaw edema
edema (mean±
(mean ± SEM)
SEM)inindifferent groups.
different (B) Inhibition
groups. of paw edema
(B) Inhibition of paw edema
in rats treated with myrtle methanolic extract. Ns, no significant difference. * p < 0.05, ** p < 0.01, ***
in rats treated with myrtle methanolic extract. Ns, no significant difference. * p
p < 0.001 (t test) using GraphPad Prism 8 for Microsoft Office. IC50 analysis performed using < 0.05, ** p < 0.01,
*** p < 0.001 (t test)
GraphPad Prismusing
8. GraphPad Prism 8 for Microsoft Office. IC50 analysis performed using
GraphPad Prism 8.
2.4.3. In Silico Study
The binding energy between molecules determines the effect of molecular docking.
Lower molecular docking-binding energy represents higher binding affinity. When the
binding energy is <5 kcal/mol, the receptor and ligand have relatively good binding prop-
erties.
The anti-inflammatory mechanism of the extract as determined by in vivo and in
Molecules 2024, 29, 849 9 of 32

The binding energy between molecules determines the effect of molecular docking.
Lower molecular docking-binding energy represents higher binding affinity. When the bind-
ing energy is <5 kcal/mol, the receptor and ligand have relatively good binding properties.
The anti-inflammatory mechanism of the extract as determined by in vivo and in vitro
experiments was demonstrated by the in silico analysis.
• Drug-likeness of Compounds
As shown in Supplementary Table S1, the compounds of the extract were evaluated
for their ability to serve as active oral drugs. Out of the 57 compounds from MMEx, 27
were considered to be drug-like compounds while the remaining 30 were not included in
further analyses, since they failed the drug-likeness test.
• Effect on pro-inflammatory targets: Cox-2, IL-1β, NF-κB, PLA2, and TNF-α
The 27 compounds were virtually screened against five key anti-inflammatory proteins:
Cox-2, IL-1β, NF-κB, PLA2, and TNF-α. Among the 27 compounds, 5 were observed to
have good binding affinity based on the docking scores: pinostrobin chalcone, cinnamyl
cinnamate, hedycaryol, totarol, and p-dimethylaminobenzophenone-. The outcome is
summarized in Table 3. The chemical structures of the five compounds are presented
Molecules 2024, 29, x FOR PEER REVIEW in
10 of 33
Figure 6.
Table 3. Docking scores of compounds and standard drugs within binding pocket of pro-
Table 3. Docking scores of compounds and standard drugs within binding pocket of pro-inflamma-
inflammatory
tory receptors. receptors.
Ligand
Ligand Docking
Docking Scores
Scores (kcal/mol)
(kcal/mol)
COX-2COX-2 IL-1βIL-1β NF-κB NF-κBPLA2PLA2 TNF-αTNF-α
Diclofenac
Diclofenac −7.7 −7.7 −6.1 −6.1 −6.1 −6.1 −7.7 −7.7 −5.7 −5.7
Pinostrobin
Pinostrobinchalcone
chalcone −8.8 −8.8 −5.7 −5.7 −5.7 −5.7 −7.4 −7.4 −6.2 −6.2
Cinnamyl cinnamate
Cinnamyl cinnamate −9.5 −9.5 −5.5 −5.5 −6.5 −6.5 −8.0 −8.0 −6.1 −6.1
Hedycaryol −7.3 −6.2 −5.8 −7.2 −6.2
Hedycaryol −7.3 −6.2 −5.8 −7.2 −6.2
Totarol −8.0 −6.2 −7.1 −8.1 −7.0
Totarol −8.0 −6.2 −7.1 −8.1 −7.0
P-dimethylaminobenzo-
−8.5
P-dimethylaminobenzophenone −8.5 −6.1 −6.1 −6.4 −6.4 −8.5 −8.5 −6.5 −6.5
phenone
Figure
Figure6.6.Chemical
Chemicalstructure of five
structure compounds
of five fromfrom
compounds myrtle methanolic
myrtle extract
methanolic that presented
extract good
that presented
binding affinity against key anti-inflammatory proteins (Cox-2, IL-1β, NF-κB, PLA2, and TNF-α).
good binding affinity against key anti-inflammatory proteins (Cox-2, IL-1β, NF-κB, PLA2, and
(A) pinostrobin chalcone; (B) cinnamyl cinnamate; (C) hedycaryol; (D) totarol; (E) p-dimethylami-
TNF-α). (A) pinostrobin chalcone; (B) cinnamyl cinnamate; (C) hedycaryol; (D) totarol; (E) p-
nobenzophenone.
dimethylaminobenzophenone.
Pinostrobin chalcone (−8.8 kcal/mol) and cinnamyl chalcone (−9.5 kcal/mol) were
found to have higher binding scores than the other compounds and diclofenac when
docked against the active site of Cox-2. For IL-1β, hedycaryol (−6.2 kcal/mol) and totarol
(−6.2 kcal/mol) stood out from the other compounds after docking. Cinnamyl cinnamate
(−6.5 kcal/mol) and totarol (−7.1 kcal/mol) were found to have good binding scores with
NF-κB. However, totarol (−8.1 and −7.0 kcal/mol) and p-dimethylaminobenzophenone
in the active site of NF-κB. The prominent amino acids interacting with the atoms of the
two hit compounds and diclofenac via hydrophobic and pi-interactions are shown in light
green (van der Waals), purple (pi-sigma), violet (pi-pi stacked), and light pink (alkyl/pi-
alkyl) (Figure 9).
The chemical interactions with diclofenac, p-cimethylamino benzophenone, and to-
Molecules 2024, 29, 849 10 of 32
tarol were primarily hydrogen bonds and hydrophobic and pi-interactions with the phos-
pholipase A2 active site (Figure 10A–C). Hydrogen bonds formed between the atoms of
p-dimethylaminobenzophenone, totarol, and the amino acid side chains of Gly29, Asp48,
Pinostrobin
His27, and Glu55 chalcone (−8.8site
in the active kcal/mol) and cinnamyl
of phospholipase A2. Thechalcone
predominant (−9.5 kcal/mol)
amino acids were
interacting
found to havewith the atoms
higher of thescores
binding two hit than
compounds and diclofenac
the other compounds via hydrophobic and when
and diclofenac
pi-interactions
docked against the areactive
shownsite
in light green For
of Cox-2. (vanIL-1β,
der Waals), purple (pi-sigma),
hedycaryol violet (pi-pi
(−6.2 kcal/mol) and totarol
stacked),
(−6.2 orange
kcal/mol) (pi-charges),
stood out from andthelight pinkcompounds
other (alkyl/pi-alkyl) (Figure
after 10). Cinnamyl cinnamate
docking.
The molecular interaction fingerprints observed with diclofenac, p-dimethylamino-
(−6.5 kcal/mol) and totarol (−7.1 kcal/mol) were found to have good binding scores with
benzophenone, and totarol after visualization were majorly hydrogen bonds, hydropho-
NF-κB. However,
bic and pi-interactions (−8.1
totarolwith and −site
the active 7.0ofkcal/mol) and11A–C).
TNF-α (Figure p-dimethylaminobenzophenone
The hydrogen bonds
(−8.5 and −
formed 6.5 kcal/mol)
between the atomswere found to be good binders oftotarol,
of p-dimethylaminobenzophenone, PLA2 and andthe
TNF-α,
amino respectively.
acid
Thechains
side molecular interaction
of Gly122, analysis
Gly121, Leu120, andofTyr59
compounds with
in the active sitediclofenac thatpredom-
of TNF-α. The were found to
haveinant
good binding
amino with the five
acids interacting withtargets
the atomsis presented incompounds
of the two hit Figures 7–11. The analysis
and diclofenac via shows
howhydrophobic
the functionaland groups
pi-interactions are shown in interact.
of the compounds light green (van interactions
These der Waals), purple (pi- to the
are critical
sigma),
stability andviolet (pi-pi of
binding stacked),
a given and light pink (alkyl/pi-alkyl)
compound in the binding (Figure
pocket 11).of a given protein.
Figure 7. Binding interaction of COX-2 with (A) diclofenac, (B) cinnamyl cinnamate, and (C) pi-
Figure 7. Binding interaction of COX-2 with (A) diclofenac, (B) cinnamyl cinnamate, and (C) pinos-
nostrobin chalcone. Purple indicates pi-sigma, violet indicates pi-pi stacked, and light pink indicates
trobinalkyl/pi-alkyl.
chalcone. Purple indicates pi-sigma, violet indicates pi-pi stacked, and light pink indicates
alkyl/pi-alkyl.
A
Molecules 2024, 29, 849 Figure 7. Binding interaction of COX-2 with (A) diclofenac, (B) cinnamyl cinnamate, and 11 (C)of pi-
32
nostrobin chalcone. Purple indicates pi-sigma, violet indicates pi-pi stacked, and light pink indicates
alkyl/pi-alkyl.
Figure 8. Binding interaction of IL-1β with (A) diclofenac, (B) hedycaryol, and (C) totarol.
A
Molecules 2024, 29, 849 12 of 32
Figure 9. Binding interaction of NF-κB with (A) diclofenac, (B) cinnamyl cinnamate, and (C) to-
Figure 9. Binding interaction of NF-κB with (A) diclofenac, (B) cinnamyl cinnamate, and (C) totarol.
tarol.
The type of interaction observed with diclofenac, cinnamyl cinnamate, and pinos-
trobin chalcone after visualization were mainly hydrogen bonds and hydrophobic and
pi-interactions with the binding site of Cox-2 (Figure 7A–C). Hydrogen bonds were estab-
lished between the atoms of cinnamyl cinnamate and pinostrobin chalcone and side chains
of Tyr385 and Leu531 while those of diclofenac were with His207 and Asn382 (Figure 7).
Molecules 2024, 29, 849
x FOR PEER REVIEW 15 of 32
13 of 33
Figure 10.10. Binding

Binding interaction
interaction of phospholipase
of phospholipase A2 with (A)A2 with (B)
diclofenac, (A)p-dimethylaminobenzo-
diclofenac, (B) p-
dimethylaminobenzophenone,
phenone, and (C) totarol. and (C) totarol.
Molecules 2024, 29, 849 14 of 32
Figure 11. Binding interaction of TNF-α with (A) diclofenac, (B) p-dimethylaminobenzophenone,
Figure
and (C) 11. Binding interaction of TNF-α with (A) diclofenac, (B) p-dimethylaminobenzophenone,
totarol.
and (C) totarol.
The predominant amino acids interacting with the atoms of the two hit compounds
and diclofenac via hydrophobic and pi-interactions are shown in light green (van der Waals).
Molecules 2024, 29, 849 15 of 32
Following visualization, hydrogen bonds, hydrophobic interactions, and pi-interactions

with the IL-1β active site were the main types of molecular interaction seen with diclofenac,
hedycaryol, and totarol (Figure 8A–C). No hydrogen bonds formed between the atoms
of totarol and the amino acid side chains in the active site of IL-1β. Hydrogen bonds
were observed between the atoms of diclofenac, hedycaryol, totarol, and side chains of
Asn66 and Leu62. The predominant amino acids interacting with the atoms of the two hit
compounds and diclofenac via hydrophobic and pi-interactions are shown in light green
(van der Waals), purple (pi-sigma), and violet (pi-pi stacked) and light pink (alkyl/pi-alkyl)
(Figure 8).
The molecular interactions observed with diclofenac, cinnamyl cinnamate, and totarol
after visualization were mainly hydrogen bonds (diclofenac, Ile298) and hydrophobic and
pi-interactions with the active site of NF-κB (Figure 9A–C). No hydrogen bonds formed
between the atoms of cinnamyl cinnamate, totarol, and the amino acid side chains in the
active site of NF-κB. The prominent amino acids interacting with the atoms of the two hit
compounds and diclofenac via hydrophobic and pi-interactions are shown in light green
(van der Waals), purple (pi-sigma), violet (pi-pi stacked), and light pink (alkyl/pi-alkyl)
(Figure 9).
The chemical interactions with diclofenac, p-cimethylamino benzophenone, and to-
tarol were primarily hydrogen bonds and hydrophobic and pi-interactions with the phos-
pholipase A2 active site (Figure 10A–C). Hydrogen bonds formed between the atoms of
p-dimethylaminobenzophenone, totarol, and the amino acid side chains of Gly29, Asp48,
His27, and Glu55 in the active site of phospholipase A2. The predominant amino acids
interacting with the atoms of the two hit compounds and diclofenac via hydrophobic and
pi-interactions are shown in light green (van der Waals), purple (pi-sigma), violet (pi-pi
stacked), orange (pi-charges), and light pink (alkyl/pi-alkyl) (Figure 10).
The molecular interaction fingerprints observed with diclofenac, p-dimethylaminoben-
zophenone, and totarol after visualization were majorly hydrogen bonds, hydrophobic and
pi-interactions with the active site of TNF-α (Figure 11A–C). The hydrogen bonds formed
between the atoms of p-dimethylaminobenzophenone, totarol, and the amino acid side
chains of Gly122, Gly121, Leu120, and Tyr59 in the active site of TNF-α. The predominant
amino acids interacting with the atoms of the two hit compounds and diclofenac via
hydrophobic and pi-interactions are shown in light green (van der Waals), purple (pi-
sigma), violet (pi-pi stacked), and light pink (alkyl/pi-alkyl) (Figure 11).
2.5. ADMET Results

The admetSAR web platform (http://lmmd.ecust.edu.cn/admetsar2; accessed on
25 June 2023) was used to predict the absorption, distribution, metabolism, and toxic-
ity (ADMET) of the reference drug (DCF) and the hit compounds (cinnamyl cinnamate,
pinostrobin chalcone, hedycaryol, totarol, and methanone [4-methyl-6-(4-dimethylamino)-
1,5,2-dioxazinan-3-yl] (phenyl)-). In the absorption class, caco-2 permeability, human
intestinal absorption, human oral availability, p-glycoprotein inhibition, and substrates
were predicted. All were predicted to permeate caco-2, which is absorbed by the human
intestine. Only DCF and hedycaryol are orally bioavailable. Methanone [4-methyl-6-(4-
dimethylamino)-1,5,2-dioxazinan-3-yl] (phenyl)-) was predicted to inhibit p-glycoprotein,
and none of the compounds can serve as a substrate for p-glycoprotein.
The distribution of these molecules was predicted via two properties, PPB and BBB.
DCF, pinostrobin chalcone, and totarol might be able to reach target sites in high to moderate
doses, as their ability to bind to plasma proteins was predicted to be high. Only pinostrobin
chalcone cannot permeate the blood–brain barrier (BBB). All compounds were predicted to
be localized in the mitochondria except hedycaryol, which is localized in the lysosome.
The metabolism of these molecules via the human liver was predicted in order to
ascertain whether they can cause harm or not. Diclofenac, cinnamyl cinnamate, pinostrobin
chalcone, totarol, and methanone [4-methyl-6-(4-dimethylamino)-1,5,2-dioxazinan-3-yl]
(phenyl)-) were predicted to inhibit cytochrome P450 1A2. Also, cinnamyl cinnamate and
Molecules 2024, 29, 849 16 of 32
pinostrobin chalcone were predicted to inhibit cytochrome P450 2C19. The inhibitors of
cytochrome P450 2C9 were predicted to be diclofenac and pinostrobin chalcone. Only
pinostrobin chalcone was predicted to inhibit cytochrome P450 3A4.
DCF and totarol were predicted to be substrates for cytochrome P450 2C9. Totarol
was also predicted to be a substrate for cytochrome P450 2D6 and 3A4. Methanone [4-
methyl-6-(4-dimethylamino)-1,5,2-dioxazinan-3-yl] (phenyl)-) was likewise predicted to be
a substrate for cytochrome P450 3A4.
All compounds were catalyzed by UGT except pinostrobin chalcone, as predicted
by admetSAR. Toxicity was evaluated based on acute oral toxicity, Ames mutagenesis,
carcinogenicity, and human hepatotoxicity. None of the compounds were predicted to
be carcinogenic, although diclofenac, pinostrobin chalcone, hedycaryol, and methanone
[4-methyl-6-(4-dimethylamino)-1,5,2-dioxazinan-3-yl] (phenyl)-) were predicted to be hepa-
totoxic. Cinnamyl cinnamate and hedycaryol were predicted to have Ames mutagenicity.
The acute oral toxicity (kg/mol) was also predicted (Table 4).
Table 4. ADMET profiling of hit compounds in relation to diclofenac according to admetSAR.
P-Dimethyl-
Cinnamyl Pinostrobin
ADMET Properties Diclofenac Hedycaryol Totarol Aminoben-
Cinnamate Chalcone
zophenone
Ames mutagenesis - + - + - -
Acute oral toxicity (c) II III III IV III III
Blood–brain barrier + + - + + +
Caco-2 + + + + + +
Carcinogenicity - - - - - -
CYP1A2 inhibition + + + - + -
CYP2C19 inhibition - + + - - -
CYP2C9 inhibition + - + - - -
CYP2C9 substrate + - - - + -
CYP2D6 inhibition - - - - - -
CYP2D6 substrate - - - - + -
CYP3A4 inhibition - - + - - -
CYP3A4 substrate - - - - + +
CYP inhibitory
- + + - - -
promiscuity
Hepatotoxicity + - + + - +
Human
ether-a-go-go-related gene - - - + - +
inhibition
Human intestinal
+ + + + + +
absorption
Human oral
+ - - + - -
bioavailability
Nephrotoxicity + - - - - -
Acute oral toxicity 2.782 1.575 2.181 1.198 3.08 2.096
P-glycoprotein inhibitor - - - - - +
P-glycoprotein substrate - - - - - -
1.01 0.767 0.955 0.779
Plasma protein binding 0.973 (97.3%) 0.852 (85.2%)
(101%) (76.7%) (95.5%) (77.9%)
Subcellular localization Mitochondria Mitochondria Mitochondria Lysosomes Mitochondria Mitochondria
UGT catalysis - - + - - -
Water solubility −4.467 −3.823 −3.1 −2.976 −4.28 −2.652
3. Discussion
3.1. Phytochemical Analysis
The good ethnopharmacological reputation of Myrtus communis L. is partly responsible
for the choice of this plant for investigation. The presence of various bioactive compounds
has justified its use from ancient times for the treatment and management of inflammation
Molecules 2024, 29, 849 17 of 32
and accompanying symptoms. Researchers have confirmed that leaf extracts have a better
antioxidant effect compared to myrtle berry extracts due to the galloyl derivatives, flavonols,
and flavonol derivatives [30], which informed the use of leaves in our work.
The resulting viscous mass of MMEx had a dark green color and a fruity, herbaceous
aroma. The extraction yield was 34.30% (wt/wt) of dry material on average, which was
similar to the 35.56% reported in extract from Zaccar, Ain Defla, Algeria [40]. Our results
partially agree with those of other reports, in which the yield was 30.6% [41]. In contrast,
our yield was lower than what was previously obtained from Zakynthos, a Greek island,
with percentages ranging between 43.4 and 59.5% [42], while it was noted to be higher
than the 28.66% reported in a study by Hayder et al. for leaves harvested from northeast
Tunisia [43]. A lower yield of 10.50% was reported in another study [44].
Our findings indicate that the phenolic content (415.85 ± 15.52 mg GAE/g DW) was
higher than the flavonoid content (285.8 ± 1.64 mg QE/g DW). This is in agreement
with the results reported in Giza, Egypt, where the TPC of myrtle alcoholic extract was
472.47 ± 3.73 mg GAE/g and the TFC was 281.15 ± 21.88 mg QE/g, which is within the
range obtained in our study. Gardeli and co-workers reported a high content of phenolics
from the methanolic extract of myrtle leaves ranging between 307 ± 7.4 and 373 ± 0.5 mg
GAE/g [42]. Bouaziz et al. [45] reported a TPC of 260.44 ± 2.52 mg GAE/g and a TFC of
26.77 ± 0.46 mg QE/g, which are lower than our results [45]. A low TPC of 33.67 mg GAE/g
was reported in a study by Aidi Wannes and his research team in Tunisia [46]. Our content
was higher when compared to that obtained with MMEx from Turkey, which had TPC
and TFC values of 190.85 ± 0.73 mg GAE/g and 43.97 ± 2.07 mg QE/g, respectively [44].
Tannins are secondary antioxidants that have the capacity to bind metal ions like Fe2+ ,
interfere with one of the stages in the Fenton reaction, and slow down the oxidation process
in addition to their anti-inflammatory, antitumor, and antimicrobial properties [47].
The total phenolic and flavonoid contents do not give a complete picture of the quality
and quantity of the phenolic and flavonoid constituents, and GC-MS analysis can provide
the most helpful information on individual phenolic compounds [48]. To our knowledge,
MMEx has not been analyzed by GC-MS in previous studies. For this reason, comparisons
were made with other species of the Myrtaceae family. Herein, 57 compounds identified
in MMEx, constituting 71% of the whole extract, are listed in the order of their column
elution time in Table 3, and they are already known for several biological activities. The
predominant compound, 1.8-cineole (33.8%), which possesses important anti-inflammatory
activity, is widely used in the cosmetic and pharmaceutical industries [49]. The second
main substance, α-pinene (10.06%), was noted to have antioxidant, anticancer, and anti-
inflammatory activities. Linalool (4.83%) has also been reported to have antioxidant,
antimicrobial, anti-inflammatory, antitumor, and insecticidal properties [25,28].
It is well known that several factors affect the yield of extraction, the kinetics of
phenolic compounds and flavonoids released from the solid matrix, and the composition
quality of the extract, such as the part of the plant used and its geographical origin, the
period of harvest, and the extraction procedure [50]. In general, the yield of the extraction
process is directly influenced by the polarity indices, types, and concentrations of organic
solvents with the extraction time [51,52].
Phytochemical analysis vividly proved that our extract is a rich source of phenolic
compounds; their presence in high concentrations explains the higher level of antioxidant
and anti-inflammatory activities. In this scope, we decided to unravel the biological
properties of MMEx using in vitro, in vivo, and in silico approaches.

The scientific community has shown heightened interest in exploring medicinal plants,
and this spike in interest can be attributed to the increasing number of publications focused
on the topic, reflecting a growing recognition of their biological value, which represents a
new wave of secure therapies for the prevention, management, and treatment of diseases
involving oxidative stress and inflammation [53]. It is believed that a single antioxi-
Molecules 2024, 29, 849 18 of 32
dant property model cannot precisely mirror the mechanism of action of an investigated
plant [54,55]; thus, modeling is frequently performed using combinations of parameters,
with a focus on the kinetics of reactions [56].
In the DPPH radical scavenging test, MMEx presented a radical quenching percent-
age greater than 80% at the highest tested concentration (200 µg.mL−1 ), with an IC50 of
33.08 ± 1.86 µg.mL−1 , indicating statistically non-significant activity when compared to
ascorbic acid (IC50 of 35.02 ± 2.57 µg.mL−1 ). This finding agrees with another study in
which the methanolic extract of Italian myrtle was found to have an inhibition percentage
greater than 90% at a concentration of 50 µg.mL−1 by DPPH testing [57]. Our results are also
in accordance with those reported in a study by Gardeli et al., in which MMEx effectively
removed DPPH with an IC50 ranging between 9.54 ± 0.93 and 17.1 ± 0.78 µg.mL−1 [42].
Excellent scavenging activity of myrtle extract, with an IC50 value of 6 µg.mL−1 , was also
reported [45].
H2 O2 is the primordial source of OH• . In the presence of transition elements in
reduced form (e.g., Fe2+ ), the Fenton and Haber–Weiss reactions lead to the formation
of the most harmful radical OH• with a limited lifespan. Its half-life in the biological
system is about 1 ns, and it reacts quickly with organic molecules with rate constants
of 109–1010 M−1 s−1 . OH• is involved in various deadly diseases such as cancer by its
ability to activate oncogenes, especially C-Raf-1 and K-ras [57,58]. Hydroxyl radicals are
especially harmful because of their ability to reduce disulfide bonds in proteins, lead-
ing to unfolding and unnatural refolding into abnormal configurations [59]. In these
circumstances, removing hydrogen peroxide is very important, since this radical is very
reactive with cell machinery [60]. In our study, MMEx displayed strong H2 O2 and hydroxyl
radical scavenging activity in a content-dependent manner, reflected by IC50 values of
17.81 ± 3.67 and 11.52 ± 0.20 µg/mL, respectively. The extract of M. communis considerably
decreased the degradation of Fe2+ via H2 O2 compared to vitamin C, with an IC50 value
of 275 µg.mL−1 [45]. Our result is more important than the one in a study by Benchikh
et al. [61], where MMEx reduced hydroxyl radicals with an IC50 of 140 µg/mL.
The ABTS and FRAP scavenging activity of MMEx was reflected by IC50 values of
22.83 ± 0.56 and 47.09 ± 4.41 µg/mL, respectively. A similar trend was observed for the
ABTS•+ removal capacity of MMEx, where the IC50 was 13.6 µg.mLl−1 [62]. In another
study, the authors reported that myrtle extract effectively scavenged ABTS•+ , with an IC50
of 4.8 µg.mL−1 [45].
The antioxidant properties of plants are frequently associated with the polyphenolic
content of aromatic amines, phenolic acids, essential oils, flavonoids, proanthocyanins, and
bioactive compounds [63,64], which can act as electron donors and react with free radicals
to transform them into more stable products and prevent radical chain reactions [65]. The
higher antioxidant power of MMEx may support the substantial amount of cell-reinforcing
chemicals present in our extract [66]. According to the literature, a phenolic content
higher than 20 mg GAE/g can be considered very high and sufficient to ensure biological
prevention against oxidants [67]. When ROS combine with the extract, a chain of events is
initiated and reactive phenoxy radicals are formed when phenol groups accept electrons,
which again combine with ROS and act as reducing agents, singlet oxygen quenchers, and
hydrogen-donators, interrupting the oxidation action chain [68]. The ability of polyphenols
to rescue the redox balance, their structural motifs, such as the carboxylic acid group, and
the presence of hydroxy and methoxy groups with lower bond dissociation energy are
responsible for their extensive biological activities [52,69,70].

3.3.1. In Vitro: Inhibition of BSA Denaturation
Given the plant’s strong antioxidant effect under examination and the close correlation
between inflammation and oxidative stress, anti-inflammatory activity was also investi-
gated in the present study using an in vitro model of protein denaturation, which, given
Molecules 2024, 29, 849 19 of 32
its features of being simple, quick, and inexpensive, represents the gold standard for the
evaluation of this parameter [71].
Inflammation has been known to be linked with protein denaturation, membrane
alteration, increased vascular permeability, and pain. Chronic inflammation has been
closely associated with tumor progression [71]. Denaturation causes changes in the physio-
chemical properties of protein, which can be caused by inflammatory agents [72]. Some
external factors, including stress and chemicals, can induce denaturation, leading to the
loss of the highly ordered quaternary, tertiary, and secondary structures and the breakage
of many weak bonds, including hydrogen bonds responsible for the native structure of
protein; this is one of the significant characteristics of inflammation. It was suggested
that bioactive substances that are capable of inhibiting the production of heat-induced
protein denaturation can be used as therapeutic anti-inflammatory drugs [55]. The search
for highly biocompatible drugs from plants is an ongoing research effort. Therefore, the
anti-inflammatory activity of MMEx was investigated by determining its potential to pro-
tect against protein denaturation. MMEx was found to be effective against heat-induced
BSA denaturation. DCF displayed a maximum inhibition of 99.73 ± 0.27% at 200 µg/mL
(Figure 3A) [73]. The data suggest that MMEx is protective against heat-induced albumin
denaturation. Further, it was found that an increased concentration of MMEx increased
the inhibition of denaturation. Extracts at a concentration of 200 µg/mL showed good
anti-inflammatory potential by inhibiting heat-induced albumin denaturation by 58.27%.
3.3.2. In Vivo: Car-Induced Paw Edema

Investigations into the anti-inflammatory potency of Myrtus communis are limited.
Only a few research reports suggest that MMEx can reduce Car-induced paw edema in
animals, although the precise mechanism is not entirely addressed. Edema is one of the
obvious indicators of inflammation and a key factor to take into account when assessing
the anti-edematogenic and anti-inflammatory properties of a substance [74]. Carrageenan
is an acknowledged pro-inflammatory agent that has been used to initiate acute inflam-
matory episodes upon injection into the footpad of rodents, and it well recapitulates the
pathogenesis characteristics [75,76]. Carrageenan has been found to resist biodegradation
due to its persistence within Kupffer cells for several months [9]. It is characterized by
the excessive generation of free radicals and inflammatory cytokines, which results in the
elevated inflammation and sensitivity of axonal terminals to pain [76]. Carrageenan causes
acute inflammation via a biphasic pattern: (1) the early phase, lasting from 1 to 2.5 h, with
the release of mediators including histamine, serotonin, 5-hydroxytryptamine, kinin, and
bradykinin; and (2) the late phase, lasting from 2.5 to 6 h, with an elevation in PGs and
COX-2, neutrophil infiltration, and ROS production [77].
In our experiment, significant paw edema in the Car-injected animals was observed in
all groups. Pretreatment with MMEx inhibited the second phase of Car-induced edema
in a dose-dependent manner relative to the positive control group. Two hours after Car
administration, there was no significant difference in the inhibition of paw edema between
animals pretreated with DCF and the two doses of MMEx. DCF inhibits PLA2, a key
mediator of several processes in the inflammatory cascade, interrupting this cycle [78]. It
was reported that NSAIDs inhibit only the late phase when PGs and Cox-2 enzymes are
detectable [79]. The same result was reported in a study by Amira et al. [80], in which
myrtle extract had the highest inhibitory activity against paw edema induced by Car (60%)
after 3 h [81]. These data are consistent with the findings of other studies, in which alcoholic
extract from myrtle leaves reduced edema by 56.6% compared to indomethacin (64.3%) [81].
The anti-inflammatory effect of Myrtus communis was investigated in several studies, and
oligomeric nonprenylated acylphloroglucinols isolated from myrtle were determined to
be potent inhibitors of eicosanoid biosynthesis [10]. Oral administration of hydroalcoholic
extract from Lampaya medicinalis Phil. in rats resulted in reduced paw edema, which was
lower compared to our result [82]. A similar trend was found in a xylene-induced ear
edema model; Hosseinzadeh et al. [28] found that ethanolic extracts of the aerial parts of
Molecules 2024, 29, 849 20 of 32
M. communis L. (50 mg.kg−1 ) effectively and significantly reduced inflammation through

the proliferative phase with an inhibition of 59.8% [28]. The ethanolic extract of myrtle at a
dose of 44.4 mg/kg was reported to inhibit edema by 34.7% [83].
Anti-inflammatory activity was evaluated by measuring 6-keto-prostaglandin F1 and
[3H]-arachidonic acid metabolite production in keratinocytes stimulated for inflammation,
and the results showed that the extract significantly decreased all metabolite production
from the pathway [25]. When administered intraperitoneally at a dose of mg/kg, M. com-
munis reduced the development of Car-induced mouse paw edema in a dose-dependent
manner [84]. Myrtucommulone (MC), semimyrtucommulone (S-MC), and nonpreny-
lated acylphloroglucinols in the leaves of M. communis were found to potently suppress
eicosanoid biosynthesis by directly inhibiting cyclooxygenase-1 and 5-lipoxygenase in vitro
and in vivo [85]. However, in another report, myrtle was believed to inhibit the cell-free
mPGES-1-mediated conversion of prostaglandin H2 (PGH2) to PGE2 [86]. Seed extract
(50 mg.kg−1 for 2 months) decreased the plasma levels of inflammatory cytokines TNF-α,
IL-8, IL-6, and IL-1β as well as erythrocyte concentration, ROS level, and lipid peroxidation,
and fortified the activity of the main antioxidant enzymes [87]. It was reported that phenolic
compounds are similar to NSAIDs in their ability to decrease the chemical mediators of
inflammation [75]. We hypothesize that the increased and more varied content of bioactive
substances in MMEx explains the higher anti-inflammatory activity by diminishing TNF-α,
IL-1β, and COX targets or by blocking the NF-κB pathway and its messengers. Flavonoids
are also known to inhibit NF-κB-dependent signaling, which is required for angiogenesis,
survival, and the proliferation of cancer cells [70]. There is still a debate regarding the
biological activities of many natural extracts and how to use them in the right way [88].
Obviously, we should take into account the short duration of the experiment and
the single exposure of the tissue to the inflammatory factor. The mechanism of action of
plant extracts cannot be quickly judged; it depends on their chemical composition, and it
is notoriously difficult to attribute biological activity to a single mechanism rather than
a cascade of reactions. MMEx can cure inflammation through a synergistic effect with
multicomponent, multi-target, and multi-pathway approaches, and to verify the above
hypothesis, we used in silico tools. Comprehending the deep molecular mechanisms
could enable the design of precise roadmaps that can lead to more successful therapeutic
solutions [89].

Prior to the molecular docking run, 57 compounds from the plant extract with anti-
inflammatory properties were subjected to drug-likeness analysis. This analysis ensured
the removal of phytocompounds with poor pharmacokinetic parameters from the drug
pipeline, thus saving time and cost. Using the SwissADME web tool, we found that
27 of the 57 screened compounds could potentially be used as drugs based on multiple
scoring schemes.
The binding affinity between the docked compounds (ligands) and target proteins in
this study were influenced by non-covalent interactions such as hydrogen bonds, hydropho-
bic bonds, and pie-stack interactions (Figures 6–10). Many researchers have confirmed that
binding affinity will be higher (binding energy will be lower) if ligands are able to form
hydrophobic interactions with appreciable numbers of hydrophobic amino acid residues in
the binding site of a target protein [90,91]. Stojanovi and Zari [92] reported the significance
of hydrophobic interactions in many systems with strong intermolecular forces [92]. This
may be responsible for the appreciable binding affinity of the active compounds docked
against the selected proteins in this study. Notably, the impact of hydrogen bonds on
the stabilization of molecular interactions between ligands and proteins should not be
downplayed due their critical role in enzyme catalysis and protein–substrate and protein–
inhibitor complexes, including the structural stability of many biological molecules [89,92].
To this end, the analysis of molecular interactions exhibited by the active compounds as-
sessed in this study as judged by their binding energy, hydrogen bonding, and hydrophobic
Molecules 2024, 29, 849 21 of 32
interactions with the surrounding amino acids in the selected protein targets revealed the
positive impact of their ligand–protein complex status, which may enhance the mecha-
nism of action of the essential oil in ameliorating chronic inflammation. The logic behind
performing the absorption, distribution, metabolism, excretion, and toxicity (ADMET)
screening of any chemical compound within the human body is that it can determine the
pharmacological and pharmacodynamic properties of candidate drug compounds within
the biological system.
Adsorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) screening
is an increasingly necessary technique for the therapeutic evaluation of small molecules
for further processing. Based on this in silico approach, more small molecules have been
discovered as potential therapeutic candidates [93]. The increasing failure rate of drug
molecules at the clinical trial stage has been attributed to poor ADME/Tox profiles. Hence,
predictions of good ADMET profiles of natural compounds can help to eliminate com-
pounds with unacceptable side effects [94]. Therefore, the in silico approach remains the
cheapest and most time-efficient model for screening bioactive molecules [95]. In this study,
the ADME/Tox and drug-likeness analysis of the phytochemicals from M. communis L. was
performed to screen out compounds with potential toxicity that violate the drug-likeness
rules. Out of the 57 phytochemicals obtained from M. communis L. via GC-MS analysis,
27 were predicted to have good pharmacokinetic profiles and obey the Lipinski, Vebers,
and Egan rules of drug-likeness.
While several rules have been developed and used to predict the drug-likeness of
bioactive compounds from plants, the most prominently used is Lipinski’s rule of five,
which was considered in this study. According to this rule, drug-like compounds should
not violate more than one of the following rules: a number of H-bond acceptors less
than 10, a number of H-bond donors less than 5, a molecular weight less than 500, a
partition coefficient less than or equal to 5, and a polar surface area less than or equal
to 14 Å [96]. The results from this study (Table 4) reveal that all the hit compounds
were positive for human intestinal absorption and Caco-2 and were negative for the P-
glycoprotein substrate, CYP2D6 inhibition, and carcinogenicity, while pinostrobin chalcone
was the only compound that was negative for blood–brain barrier penetration. Most orally
administered drugs exhibit their potency when they are absorbed into the bloodstream
for circulation [97]. The ability of bioactive molecules to cross the blood–brain barrier
is a significant consideration for the management of neurodegenerative disorders [98].
Human intestinal absorption and Caco-2 remain as the tools for screening the intestinal
permeability of therapeutic agents. Hence, the hit compounds in this study can effectively
permeate the intestine for metabolism. The membrane P-glycoprotein (P-gp) is known to
inhibit the absorption, distribution, and bioavailability of its substrate as small molecules,
thereby eliminating them from circulation [93]. The negative result predicted for the hit
compounds revealed that they have high bioavailability in the system. The hit compounds
were also revealed to be non-carcinogenic, indicating the safety of the plant.
Virtual screening via molecular docking is an in silico model that employs a computer-
based tool that has been used in structure-based drug development [93]. It relies on
the principle of predicting the binding orientation and evaluating the binding energy of
small molecules interacting at the binding pocket of the target. The results of molecular
docking presented in Table 4 show better binding affinity of the hit compounds against the
inflammatory protein. The hit compounds showed better binding affinity against COX-2
and PLA-2 compared with the standard drug (diclofenac). Hence, these proteins might
be predicted as the mechanism of anti-inflammatory action, which corroborates with the
in vitro and in vivo anti-inflammatory results obtained in this study.
Molecules 2024, 29, 849 22 of 32
4. Materials and Methods

4.1. Chemicals, Reagents, and Equipment
All reagents and solvents used in this study were of analytical grade. The 1,1-diphenyl-
2-picrylhydrazyl (DPPH) and 2,2′ -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)
free radicals and carrageenan were purchased from Sigma-Aldrich (St. Louis, MO, USA).
4.2. Collection of Plant Material

Freshly harvested plant materials (myrtle leaves) were randomly collected in the early
morning in April 2022 in Taxana, Jijel, Algeria, and identified as Myrtus communis L. The
region is located 22 km southeast of Jijel city (latitude: 36◦ 39′ 38′′ ; longitude: 5◦ 47′ 28′′ ;
altitude: 750 m).
4.3. Methanolic Extract Preparation

Methanolic extract from leaves of M. communis was prepared according to the protocol
reported by Dib et al. [99] with slight modifications. Samples were first visually examined
for any infection, damage, discoloration, or distortion. Subsequently, to remove all im-
purities, the plant specimens were subjected to a triple-washing process using tap water
followed by distilled water, then shade-dried for 2 weeks at room temperature (37 ◦ C). The
dried specimens were weighed and pulverized with a grinding machine. The fine powder
samples were preserved in air-tight canisters shielded from light until they were utilized
for subsequent procedures. The powder was subjected to extraction by maceration in 96%
methanol for 24 h with a ratio of 10:1 (v/w) of solvent. The extract was then filtered using
Wattman filter paper No. 3 to remove plant debris. Samples were centrifuged to obtain
a better quality filtrate. A rotary evaporator (BUCHI Rotavapor R-300, BÜCHI, Flawil,
Switzerland) was used to remove the methanol from the extract at 45 ◦ C with rotations of
95 to 100 rpm in vacuum. After evaporation, some parts of the extract were stored in an
Eppendorf tube at 4 ◦ C for further studies, and the remaining extract was further diluted,
filtered, and stored for phytochemical analysis. The extraction yield (EY) is expressed as a
percentage of the weight of the obtained dried extract relative to the initial dried matter of
the sample used for extraction, as described in the following equation:
weight of dry extract

% EY = × 100
weight taken for extraction
4.4. Gas Chromatography–Mass Spectrometry (CG-MS) Analysis

The phytochemical investigation of the extract was performed using a Shimadzu
QP2010 device. For the MMEx sample, the working solution (1 µL) was injected in splitless
mode and the temperature program was set as follows: initial temperature at 70 ◦ C for
4 min, then raised and maintained at 240 ◦ C and fitted with a fused-silica SE30 capillary
column (length 25 m, internal diameter 0.25 mm, film thickness 0.25 µm). The total run
time was 30.25 min. Helium (He) with a purity of 99.99% was selected as an inert carrier
gas at a flow rate of 1.4 mL/min. The ion source temperature was set at 200 ◦ C and the
interface temperature at 250 ◦ C. The electron impact ionization (EI) was 70 eV and mass
spectra were scanned in the range of 40–450 m/z. Bioactive ingredients in MMEx were
identified and authenticated based on the retention time (Rt) with respect to their spectra,
then classified by matching the spectral patterns attained with the existing NIST05 2010
mass spectral library. Detected compounds with >90% spectral matching quality were
considered acceptable. The determination of the percentage of each component was based
on peak area.
4.5. Total Polyphenol Content (TPC)

The Folin–Ciocalteu procedure was applied as previously described by Siddiqui
et al. [100]. In this procedure, 500 µL of MMEx (1.0 mg/mL) diluted by 1/100th in 2.25 mL of
distilled water was treated with 250 µL of 10% Folin–Ciocalteu reagent. After 5 min, 2 mL of
Molecules 2024, 29, 849 23 of 32
7.5% w/v sodium carbonate (Na2 CO3 ) was added. The mixture was vortexed for 15 s before
being kept at ambient temperature for 1 h to allow for color development. The interaction
between the Folin–Ciocalteu and phenolic residues led to the formation of a complex blue
color, with its intensity proportional to the concentration of polyphenols in the extract.
Using a spectrophotometer, the extract’s absorbance at 765 nm was detected. The TPC of
the extract was calculated using a curve made with gallic acid concentrations ranging from
0 to 400 mg/L; the result is represented as mg of sample gallic acid equivalent (GAE)/g.
4.6. Total Flavonoid Content (TFC)

The TFC content was determined using the method outlined by Dewanto et al. [101].
Specifically, 250 µL of the extract (1 mg/mL) was combined with 75 µL of NaNO2 (5%).
After resting for 6 min at room temperature away from light, 150 µL of AlCl3 (2%) and
500 µL of NaOH (1 M) were added, the volume was completed with 2.5 mL of distilled
water, and then a spectrophotometer was used to test the mixture’s absorbance at 510 nm.
A standard curve with quercetin concentrations ranging from 0 to 200 µg/mL was used to
determine TFC, and the results were expressed as milligrams of quercetin equivalent per
gram of dry matter (mg QE/g).
4.7. Total Hydrolyzable and Condensed Tannin Content (THT, TCT)

HT content was determined according to the protocol of Kardel et al. [102]. For this
procedure, 0.2 g of each ground organ was macerated for 18 h in 10 mL of 96% methanol,
and the mixture was filtered using Whatman No. 1 paper. Then, 1 mL of the filtrate was
added to 3.5 mL of a solution prepared based on 10−2 M ferric trichloride (FeCl3 ) in 10−3 M
hydrochloric acid (HCl). After 15 s, the absorbance of the mixture was read at 660 nm. HT
was expressed by the following formula:
A.P.M.V.FD
HT(%) =
E mole. P
where A is absorbance; E mole is 2169 for gallic acid (constant expressed in moles); M
is mass (=300); V is the volume of the extract used; PM is the molecular weight of gallic
acid (170.12 g/mol); P is the weight of the sample used; and FD is the factor of dilution.
The results, represented in the form of percentages (%), were converted into milligram-
equivalent gallic acid (EGA) per 100 g of dry matter (mg EGA/100 g MS) [98].
Condensed tannins (CTs) were quantified using the protocol of Martin et al. [103].
For this procedure, 0.2 g of each crushed organ was macerated for 18 h in 10 mL of 96%
methanol. The mixture was filtered using Whatman No. 1 paper. Then, 1 mL of filtrate was
added to 2 mL of a solution prepared in 1% vanillin base in 70% sulfuric acid. The entire
mixture was placed in a water bath for 15 min at 20 ◦ C away from light. The absorbance of
the mixture was read at 500 nm. CT content is expressed by the following formula:
A.V
CT (%) = 5.2 × 10−2 .
P
where 5.2 × 10−2 is the cyanidine-equivalent constant, A is absorbance, V is the volume

of extract used, and P is the weight of the sample. CT is expressed in % or milligram-
equivalent cyanidine (EC) per 100 g of dry matter (mg EGA/100 g MS).

The antioxidant activity of the methanolic extract of M. communis was determined
by using 5 complementary in vitro assays: DPPH• , hydrogen peroxide (H2 O2 ), hydroxyl
radical (OH• ), 2,2′ -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+ ), and ferric-
reducing antioxidant power (FRAP). This enables a more straightforward and direct com-
parison of antioxidant activity. Ascorbic acid was used as a reference standard. Six dilutions
were performed to obtain concentrations from 0 to 200 µg.mL−1 . Antioxidant activity was
quantified in terms of half-maximal inhibitory concentration (IC50 ), which generally pro-
Molecules 2024, 29, 849 24 of 32
vides outstanding potency information in pharmacological research by indicating how

much of the drug is needed to inhibit a biological process by half. A lower IC50 value
indicates a stronger ability to scavenge free radicals. All measurements were conducted in
triplicate (n = 3).
4.8.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Scavenging Assay

The antioxidant potential of MMEx was determined by DPPH free radical-capturing
assay using a procedure described by Cosmulescu et al. [104], with some amendments. In
brief, 0.5 mL of sample was mixed with 1.5 mL of DPPH-MeOH (0.2 mmol.L−1 ). This was
vigorous shaken to obtain a uniform dispersion and kept aside for 30 min in the dark at
37 ◦ C to achieve a steady state of scavenging. The active compounds manifested as yellow
marks surrounded by purple. Optical density was recorded at a wavelength of 517 nm.
More radical scavenging activity is indicated by a lower degree of absorption. DPPH in
methanol solution was used as a control. The inhibition percentage was calculated using
Equation (1):
OD0 − OD1
I (%) = × 100 (1)
OD0
where OD0 is the optical density of the control and OD1 is the optical density of the sample.
4.8.2. H2 O2 Hydrogen Peroxide Scavenging Assay

This assay was conducted as described by Khan et al. [105]. Briefly, a 40 mM solution
of hydrogen peroxide (30%) was prepared in phosphate buffer (PBS) (0.1 M; pH = 7.4).
Then, 2 mL of MMEx was added to 1.5 mL of H2 O2 solution. Using a spectrophotometer,
the absorbance of the mixture was recorded after 10 min at 230 nm. An extra blank sample
without hydrogen peroxide was prepared for background inference. The percentage of
H2 O2 inhibition was calculated using Equation (1).
4.8.3. Hydroxyl Radical (OH•) Scavenging Test

This test was performed according to the typical methodology described by Brands
et al. [106] as follows: 1 mL of MMEx was added to the reaction mixture, which consisted of
1 mL of FeSO4 (1.5 mM), 0.7 mL of H2 O2 (6 mM), and 0.3 mL of sodium salicylate (20 mM).
After 1 h of incubation at 37 ◦ C, the absence of the hydroxylated salicylate complex was
measured at 562 nm. The hydroxyl radical scavenging capacity was calculated using
Equation (1).
4.8.4. ABTS• Radical Scavenging Assay

According to the protocol described by Dong et al. [107]., the ABTS discoloration
technique initially involved converting 7 mM ABTS (colorless) to ABTS+ (blue) by adding
2.45 mM K2 S2 O8 . The prepared mixture was left in the dark at room temperature for 12 to
16 h before further use. The resulting solution was diluted in methanol in order to adjust
the absorbance at 734 nm to 0.70 ± 0.02 units in ethanol. Then, 1 mL of newly prepared
ABTS•+ solution was added to 1 mL of sample solution at various concentrations, and the
absorbance was recorded 10 min after the initial mixing. Using the same formula as the
DPPH test (Equation (1)), the percentage of inhibition was determined.
4.8.5. Ferric-Reducing Antioxidant Power (FRAP)

The ability of MMEx to reduce the ferric iron (Fe3+ ) present in the K3 Fe (CN)6 complex
to ferrous iron (Fe2+ ) was tested following the experimental protocol of Suseela et al. [108]
with minor changes. For this procedure, 0.5 mL of MMEx or ascorbic acid was mixed with
2.5 mL of PBS (0.2 M; pH 6.6) and 2.5 mL of hexacyanoferrate ([K3 Fe (CN)6 ]) solution at 1%
and incubated at 50 ◦ C for 20 min using a water bath. This step was necessary to reduce
ferricyanide into ferrocyanide. Then, 2.5 mL of 10% trichloroacetic acid was added to stop
the reaction. The tubes were centrifuged at 3000 rpm for 10 min. Then, 2.5 mL of the upper
layer of the mixture was separated and mixed with 2.5 mL of distilled water. Finally, 500 µL
Molecules 2024, 29, 849 25 of 32
of freshly prepared ferric chloride solution (0.1%) was added to the mixture to develop
the color. The absorbance at 700 nm was read against a blank after 10 min of incubation at
room temperature. The antioxidant activity and IC50 were determined.

4.9.1. In Vitro: Inhibition of Bovine Serum Albumin (BSA) Denaturation
Inhibition of albumin denaturation was performed to analyze the anti-inflammatory
activity of MMEx using a modified version of a method described by Kar et al. [109].
Diclofenac sodium (DCF) was used as a standard. The test consisted of preparing the
reaction mixture containing 2.5 mL of PBS (pH = 6.4), 0.5 mL of 5% (w/v) BSA solution, and
50 µL of the MMEx at different concentrations (200, 100, 50, 25, 12.5, 0 µg.mL−1 ). Samples
were taken out after incubation at 37 ◦ C for 15 min and then re-incubated at 70 ◦ C for 5 min
to denature the protein. After cooling, the absorbance was read at 600 nm. The following
equation was used to determine the percentage of protein denaturation inhibition:
Ac − At
I (%) = × 100
Ac
where Ac is absorbance of the control and At is absorbance of the tested sample. The
control represents the 100% denatured protein, and the results were compared with di-
clofenac sodium.
4.9.2. In Vivo: Carrageenan-Induced Paw Edema in Rats

• Animals
The experiment was conducted using female albino Wistar rats (weighing 190 ± 10.0 g)
procured from the Pasteur Institute (Algeria). A maximum of 5 rats were housed in each
cage, and each animal was given unrestricted access to standard water and pellet food.
Every day, the light/dark cycle, temperature, and humidity were maintained (22 ± 2 ◦ C,
55 ± 10%). Before the experiments, rats were acclimatized to laboratory conditions for
7 days.
• Ethical statement
The experimental procedure was compliant with internationally accepted protocols
and the ethics of laboratory animal care and use as approved by a committee of the Algerian
Association of Sciences in Animal Experimentation (No. 8808/1988), and associated with
veterinary medical activities and animal-health protection (No. JORA:004/1988).
• Experimental design
Before starting, the rats were fasted for 18 h but had free access to water the entire time.
According to the protocol of Karim et al. [110] with slight modifications, a single freshly
prepared dose of DCF (50 mg.kg−1 ), used as the reference drug, and 2 low doses of MMEx
(25–50 mg.kg−1 ) were given to rats orogastrically [29]. Then, 30 min after administration
of the appropriate experimental substance, 1% (w/v) sterile Car dissolved in 0.9% saline
solution was administered as a single 0.1 mL injection under the plantar aponeurosis of
the right hind footpad of animals in all groups. The basal thickness of the animals was
measured just before starting the induction of inflammation (time 0) and then after Car
injection at hourly intervals for 4 h, using an electronic digital Vernier caliper. The difference
between initial and successive readings represented the rate of inflammation or volume of
edema and the percentage of inhibition was measured.
4.9.3. In Silico
• Ligand Preparation
The 57 compounds were subjected to drug-likeness screening based on the Lipin-
ski, Ghose, Veber, Egan, and Muegge rules, and the bioavailability of the compounds
was determined using the SwissADME website (swissadme.ch). The 3D structures of
Molecules 2024, 29, 849 26 of 32
the 27 compounds and the reference drug diclofenac were downloaded from the NCBI
PubChem Compounds database (https://www.pubchem.ncbi.nm.nih.gov/) [111] in SDF
format. Using Discovery Studio, the SDF files were saved in PDB format.
• Protein Preparation
The target proteins, COX-2 (5F19), IL-1β (4G6M), NF-κB (1NFI), PLA2 (1DCY), and
TNF-α (2AZ5), with crystallographic resolutions of 2.04, 1.81, 2.70, 2.70, and 2.10 Å, respec-
tively, were downloaded from the Protein Data Bank (http://rcsb.org) [112]. The target
proteins were minimized and constructed using UCSF Chimera software (v 1.16). The
binding sites of proteins were predicted using BIOVIA Discovery Studio and published
studies [113,114].
• Molecular Docking Analysis
Molecular docking analysis was performed and binding affinity was determined using
Auto Dock v. 4.2 [115]. The protein targets and ligands in pdbqt format were dragged into
their respective columns. All ligands were docked, and a maximum exhaustiveness of 8 was
computed for all of them. All other parameters in the software were in default mode. The
binding affinity of compounds for the protein targets was recorded. The compounds
were then ranked by their docking scores. Also, the molecular interactions between
protein targets and compounds were viewed using BIOVIA Discovery Studio. Amino acids
engaging with the ligand, hydrogen bonds (H-bonds), hydrophobic interactions, and the
individual atoms involved were examined in each ligand cluster (Supplementary Table S2).
4.10. In Silico Pharmacokinetic Profile and Drug-Likeness Prediction

The admetSAR web platform (http://lmmd.ecust.edu.cn/admetstar2 (accessed on 29
January 2024)) was used to predict the absorption, distribution, metabolism, and toxicity
(ADMET) of the reference drug (DCF) and the hit compounds. The 57 compounds of MMEx
were subjected to drug-likeness testing based on the Lipinski, Ghose, Veber, Egan, and
Muegge rules and their bioavailability. A total of 30 compounds were screened out, leaving
27 compounds.
5. Data Analysis
Data obtained from the laboratory experiment were subjected to one-way analysis of
variance (ANOVA) followed by a post hoc LSD test, with a level of significance of p < 0.05,
using GraphPad Prism 8. All of the results were presented as the mean ± standard deviation.
6. Conclusions
After the characterization and quantification of methanolic extract from Myrtus commu-
nis L., the obtained outputs highlight that myrtle leaves are particularly rich in a plethora
of phenolic compounds, with acceptable pharmacokinetics, promising anti-inflammatory
activity, substantial antioxidant potential, and excellent safety, paving the way for an excit-
ing new chemical entity. According to these data, we believe that the investigated MMEx
could be considered to be an alternative agent for the treatment of chronic inflammation
instead of NSAIDs, as their anti-inflammatory potency is comparable, but they do not
produce the acute side effects of DCF. It would also be interesting to test a larger range
of extract concentrations to determine the lowest concentration that would maintain the
anti-inflammatory effects. For this purpose, well-designed animal and clinical studies are
needed to verify the clinical benefits of this plant extract over the long term. It is necessary
to guarantee the quantifiable and uniform content of active substances in phyto-medicines
to ensure the desired pharmacological effect and the pharmacokinetic behavior, and to
establish doses and therapeutic regimens with reduced adverse effects.
Molecules 2024, 29, 849 27 of 32
Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/molecules29040849/s1. Table S1. Druglikeness of Compounds from plants
using SwissAdme Server. Table S2. The grid center, dimensions of each target proteins and the amino
acid residue present at the active site.
Author Contributions: Conceptualization, S.B., E.L. and W.K.; methodology, S.B, W.K., E.L. and Y.B.;
software, T.V.A., H.I.U. and D.A.O.; validation, A.A.M., A.H.A. and A.A.A.; formal analysis, S.B.,
W.K. and Y.B.; investigation, S.B. and W.K.; resources, A.A.A. and Y.B.; data curation, S.B., W.K. and
M.A.O.; writing—original draft preparation, S.B. and W.K.; writing—review and editing, S.B., W.K.,
E.L. and Y.B.; visualization, S.B., W.K., E.L. and Y.B.; supervision, E.L.; funding acquisition, A.A.A.
and Y.B. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Deanship of Scientific Research at King Khalid University
under grant number RGP2/454/44. The APC was funded by King Khalid University.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article and Supplementary Materials.
Acknowledgments: The authors extend their appreciation to the Deanship of Scientific Research at
King Khalid University for funding this work through a large group research project under grant
number RGP2/454/44.
Conflicts of Interest: The authors declare no conflicts of interest.
References
1. Quinty, V.; Nasreddine, R.; Colas, C.; Launay, A.; Nehmé, R.; El-Khiraoui, A.; Piot, C.; Draye, M.; Destandau, E.; Da Silva, D.; et al.
Antioxidant and anti-lipase capacities from the extracts obtained from two invasive plants: Ambrosia artemisiifolia and Solidago
canadensis. Food Biosci. 2023, 55, 103069. [CrossRef]
2. Theodosis-Nobelos, P.; Papagiouvannis, G.; Rekka, E.A. Ferulic, Sinapic, 3, 4-Dimethoxycinnamic Acid and Indomethacin
Derivatives with Antioxidant, Anti-Inflammatory and Hypolipidemic Functionality. Antioxidants 2023, 12, 1436. [CrossRef]
[PubMed]
3. Chaudhary, M.R.; Chaudhary, S.; Sharma, Y.; Singh, T.A.; Mishra, A.K.; Sharma, S.; Mehdi, M.M. Aging, oxidative stress
and degenerative diseases: Mechanisms, complications and emerging therapeutic strategies. Biogerontology 2023, 24, 609–662.
[CrossRef] [PubMed]
4. Ramos-González, E.J.; Bitzer-Quintero, O.K.; Ortiz, G.; Hernández-Cruz, J.J.; Ramírez-Jirano, L.J. Relationship between inflamma-
tion and oxidative stress and its effect on multiple sclerosis. Neurología 2021, in press.
5. Bourais, I.; Elmarrkechy, S.; Taha, D.; Badaoui, B.; Mourabit, Y.; Salhi, N.; Alshahrani, M.M.; Al Awadh, A.A.; Bouyahya, A.;
Goh, K.W. Comparative Investigation of Chemical Constituents of Kernels, Leaves, Husk, and Bark of Juglans regia L., Using
HPLC-DAD-ESI-MS/MS Analysis and Evaluation of Their Antioxidant, Antidiabetic, and Anti-Inflammatory Activities. Molecules
2022, 27, 8989. [CrossRef] [PubMed]
6. Alam, M.M.; Emon, N.U.; Alam, S.; Rudra, S.; Akhter, N.; Mamun, M.M.R.; Ganguly, A. Assessment of pharmacological activities
of Lygodium microphyllum Cav. leaves in the management of pain, inflammation, pyrexia, diarrhea, and helminths: In vivo, in vitro
and in silico approaches. Biomed. Pharmacother. 2021, 139, 111644. [CrossRef] [PubMed]
7. Sarkar, D.; Bhattacharyya, S. Chapter 6—The phospholipase A2 superfamily and its role in chronic inflammatory conditions with
relation to adjuster cells. In Phospholipases in Physiology and Pathology; Chakraborti, S., Ed.; Academic Press: Cambridge, MA, USA,
2023; pp. 1–21.
8. Serhan, C.N.; Sulciner, M.L. Resolution medicine in cancer, infection, pain and inflammation: Are we on track to address the next
Pandemic? Cancer Metastasis Rev. 2023, 42, 13–17. [CrossRef] [PubMed]
9. Ahmed, R.F.; Radwan, O.K.; Bakeer, R.M. Resveratrol in combination with Ibuprofen against acute carrageenan-induced
inflammation and hepatic insult: Rectification of adenylate energy charge (AEC), anti-apoptotic, cell proliferation and DNA
preservation potentials. Int. J. PharmTech Res. 2016, 9, 917–938.
10. Elgazar, A.A.; Knany, H.R.; Ali, M.S. Insights on the molecular mechanism of anti-inflammatory effect of formula from Islamic
traditional medicine: An in-silico study. J. Tradit. Complement. Med. 2019, 9, 353–363. [CrossRef]
11. Gholizadeh, M.; Khalili, A.; Roodi, P.B.; Saeedy, S.A.G.; Najafi, S.; Keshavarz, M.M.; Djafarian, K. Selenium supplementation
decreases CRP and IL-6 and increases TNF-alpha: A systematic review and meta-analysis of randomized controlled trials. J. Trace
Elem. Med. Biol. 2023, 79, 127199. [CrossRef]
12. Loi, F.; Córdova, L.A.; Pajarinen, J.; Lin, T.H.; Yao, Z.; Goodman, S.B. Inflammation, fracture and bone repair. Bone 2016, 86,
119–130. [CrossRef]
Molecules 2024, 29, 849 28 of 32
13. El-Shitany, N.A.; Eid, B.G. Icariin modulates carrageenan-induced acute inflammation through HO-1/Nrf2 and NF-kB signaling
pathways. Biomed. Pharmacother. 2019, 120, 109567. [CrossRef]
14. Abdulkhaleq, L.A.; Assi, M.A.; Abdullah, R.; Zamri-Saad, M.; Taufiq-Yap, Y.H.; Hezmee, M.N.M. The crucial roles of inflammatory
mediators in inflammation: A review. Vet. World 2018, 11, 627–635. [CrossRef]
15. Panchal, N.K.; Swarnalatha, P.; Prince, S.E. Trichopus zeylanicus ameliorates ibuprofen inebriated hepatotoxicity and enteropathy:
An insight into its modulatory impact on pro/anti-inflammatory cytokines and apoptotic signaling pathways. Inflammopharmacol-
ogy 2022, 30, 2229–2242. [CrossRef]
16. Talaj, J.A.; Zielinski, K.; Bujacz, A.J. Structural Investigation of Diclofenac Binding to Ovine, Caprine, and Leporine Serum
Albumins. Int. J. Mol. Sci. 2023, 24, 1534. [CrossRef]
17. Panchal, N.K.; Sabina, E. Non-steroidal anti-inflammatory drugs (NSAIDs): A current insight into its molecular mechanism
eliciting organ toxicities. Food Chem. Toxicol. 2023, 172, 113598. [CrossRef]
18. D’antongiovanni, V.; Antonioli, L.; Benvenuti, L.; Pellegrini, C.; Di Salvo, C.; Calvigioni, M.; Panattoni, A.; Ryskalin, L.; Natale, G.;
Banni, S. Use of Saccharomyces boulardii CNCM I-745 as therapeutic strategy for prevention of nonsteroidal anti-inflammatory
drug-induced intestinal injury. Br. J. Pharmacol. 2023, 180, 3215–3233. [CrossRef]
19. Teo, S.H.; Tan, N.C.; Choo, J.C.J.; Kwek, J.L.; Kadir, H.B.A.; Bee, Y.M.; Huang, H.; Kaushik, M.; Ang, A.T.W.; Lim, C.C. Non-
steroidal anti-inflammatory drugs in chronic kidney disease and risk of acute adverse kidney events according to route of
administration. Int. Urol. Nephrol. 2023, 55, 679–686. [CrossRef] [PubMed]
20. Awan, S.; Siddiqui, K.; Munawar, M.; Inayat, S.; Shehzadi, S.; Zahid, M.A. Recent developments in the understanding of
NSAID-induced liver fibrosis: Linking fundamental mechanisms to specific therapy ideas. Int. J. Nat. Med. Health Sci. 2023, 2,
39–44.
21. Paniagua-Pérez, R.; Sánchez-Chapul, L.; Madrigal-Bujaidar, E.; Álvarez-González, I.; Madrigal-Santillán, E.; Cruz-Hernández,
L.; Martínez-Canseco, C.; Reyes-Legorreta, C.; Ruiz-Rosano, L.; Hernández-Flores, C.; et al. Anti-Inflammatory Potential of
Pteropodine in Rodents. Metabolites 2023, 13, 907. [CrossRef] [PubMed]
22. Noor, F.; Tahir, U.; Qamar, M.; Ashfaq, U.A.; Albutti, A.; Alwashmi, A.S.; Aljasir, M.A. Network pharmacology approach for
medicinal plants: Review and assessment. Pharmaceuticals 2022, 15, 572. [CrossRef] [PubMed]
23. Nyalo, P.; Omwenga, G.; Ngugi, M. Quantitative Phytochemical Profile and In Vitro Antioxidant Properties of Ethyl Acetate
Extracts of Xerophyta spekei (Baker) and Grewia tembensis (Fresen). J. Evid.-Basesd Integr. Med. 2023, 28. [CrossRef]
24. Okaiyeto, K.; Kerebba, N.; Rautenbach, F.; Singh, S.K.; Dua, K.; Oguntibeju, O.O. UPLC-ESI-QTOF-MS phenolic compounds
identification and quantification from ethanolic extract of Myrtus communis ‘Variegatha’: In vitro antioxidant and antidiabetic
potentials. Arab. J. Chem. 2023, 16, 104447. [CrossRef]
25. Alipour, G.; Dashti, S.; Hosseinzadeh, H. Review of Pharmacological Effects of Myrtus communis L. and its Active Constituents.
Phytother. Res. 2014, 28, 1125–1136. [CrossRef]
26. Dabbaghi, M.M.; Fadaei, M.S.; Soleimani Roudi, H.; Baradaran Rahimi, V.; Askari, V.R. A review of the biological effects of Myrtus
Communis. Physiol. Rep. 2023, 11, e15770. [CrossRef]
27. Mahboubi, M. Effectiveness of Myrtus communis in the treatment of hemorrhoids. J. Integr. Med. 2017, 15, 351–358. [CrossRef]
28. Hosseinzadeh, H.; Khoshdel, M.; Ghorbani, M. Antinociceptive, Anti-inflammatory Effects and Acute Toxicity of Aqueous and
Ethanolic Extracts of Myrtus communis L. Aerial Parts in Mice. J. Acupunct. Meridian Stud. 2011, 4, 242–247. [CrossRef]
29. Khosropour, P.; Sajjadi, S.E.; Talebi, A.; Minaiyan, M.J. Anti-inflammatory effect of Myrtus communis hydroalcoholic extract and
essential oil on acetic acid-induced colitis in rats. J. Rep. Pharm. Sci. 2019, 8, 204–210.
30. Hennia, A.; Miguel, M.G.; Nemmiche, S. Antioxidant Activity of Myrtus communis L. and Myrtus nivellei Batt. & Trab. Extracts: A
Brief Review. Medicines 2018, 5, 89. [PubMed]
31. Barhouchi, B.; Menacer, R.; Bouchkioua, S.; Mansour, A.; Belattar, N. Compounds from myrtle flowers as antibacterial agents and
SARS-CoV-2 inhibitors: In-vitro and molecular docking studies. Arab. J. Chem. 2023, 16, 104939. [CrossRef] [PubMed]
32. Belahcene, S.; Kebsa, W.; Omoboyowa, D.A.; Alshihri, A.A.; Alelyani, M.; Bakkour, Y.; Leghouchi, E. Unveiling the Chemical
Profiling Antioxidant and Anti-Inflammatory Activities of Algerian Myrtus communis L. Essential Oils, and Exploring Molecular
Docking to Predict the Inhibitory Compounds against Cyclooxygenase-2. Pharmaceuticals 2023, 16, 1343. [CrossRef] [PubMed]
33. Fazeli-Nasab, B.; Sayyed, R.Z.; Sobhanizadeh, A. In Silico Molecular Docking Analysis of α-Pinene: An Antioxidant and
Anticancer Drug Obtained from Myrtus communis. Int. J. Cancer Manag. 2021, 14, e89116. [CrossRef]
34. Hayder, N.; Abdelwahed, A.; Kilani, S.; Ammar, R.B.; Mahmoud, A.; Ghedira, K.; Chekir-Ghedira, L. Anti-genotoxic and
free-radical scavenging activities of extracts from (Tunisian) Myrtus communis. Mutat. Res. 2004, 564, 89–95. [CrossRef] [PubMed]
35. Moura, D.; Vilela, J.; Saraiva, S.; Monteiro-Silva, F.; De Almeida, J. Antimicrobial Effects and Antioxidant Activity of Myrtus
communis L. Essential Oil in Beef Stored under Different Packaging Conditions. Food 2023, 12, 3390. [CrossRef] [PubMed]
36. Al-Maharik, N.; Jaradat, N.; Al-Hajj, N.; Jaber, S. Myrtus communis L.: Essential oil chemical composition, total phenols and
flavonoids contents, antimicrobial, antioxidant, anticancer, and α-amylase inhibitory activity. Chem. Biol. Technol. Agric. 2023,
10, 41. [CrossRef]
37. Li, J.; Chang, R.Y.; Chen, L.F.; Qian, S.H.; Wang, R.Y.; Lan, J.L.; Huang, L.; Ding, X.H. Potential Targets and Mechanisms of Jiedu
Quyu Ziyin Decoction for Treating SLE-GIOP: Based on Network Pharmacology and Molecular Docking. J. Immunol. Res. 2023,
28, 8942415. [CrossRef] [PubMed]
Molecules 2024, 29, 849 29 of 32
38. Biswas, S.; Mita, M.A.; Afrose, S.; Hasan, M.R.; Islam, M.T.; Rahman, M.A.; Ara, M.J.; Chowdhury, M.B.A.; Meem, H.N.;
Mamunuzzaman, M.; et al. Integrated Computational Approaches for Inhibiting Sex Hormone-Binding Globulin in Male
Infertility by Screening Potent Phytochemicals. Life 2023, 13, 476. [CrossRef] [PubMed]
39. Rufa’i, F.A.; Baecker, D.; Mukhtar, M.D. Phytochemical Screening, GC-MS Analysis, and Evaluating In Vivo Antitrypanosomal
Effects of a Methanolic Extract of Garcinia kola Nuts on Rats. Antibiotics 2023, 12, 713. [CrossRef]
40. Touaibia, M.; Chaouch, F.Z. Pouvoir antioxydant des extraits de Myrtus communis L. obtenus in situ et in vitro. Nat. Technol. 2014,
7, 3–9.
41. Bouchenak, O.; Yahiaoui, K.; Benhabyles, N.; Laoufi, R.; Toubal, S.; El Haddad, D.; Oussaid, S.; Blizak, D.; Arab, K. Criblage
phytochimique et évaluation du pouvoir antioxydant des feuilles de Myrtus communis L. et Rhamnus alaternus L. Rev. Agrobiol.
2020, 10, 1749–1761.
42. Gardeli, C.; Vassiliki, P.; Athanasios, M.; Kibouris, T.; Komaitis, M. Essential oil composition of Pistacia lentiscus L. and Myrtus
communis L.: Evaluation of antioxidant capacity of methanolic extracts. Food Chem. 2008, 107, 1120–1130. [CrossRef]
43. Hayder, N.; Skandrani, I.; Kilani, S.; Bouhlel, I.; Abdelwahed, A.; Ammar, R.B.; Mahmoud, A.; Ghedira, K.; Chekir-Ghedira, L.
Antimutagenic activity of Myrtus communis L. using the Salmonella microsome assay. S. Afr. J. Bot. 2008, 74, 121–125. [CrossRef]
44. Tumen, I.; Senol, F.S.; Orhan, I.E. Inhibitory potential of the leaves and berries of Myrtus communis L. (myrtle) against enzymes
linked to neurodegenerative diseases and their antioxidant actions. Int. J. Food Sci. Nutr. 2012, 63, 387–392. [CrossRef] [PubMed]
45. Bouaziz, A.; Abdalla, S.; Baghiani, A.; Charef, N. Phytochemical analysis, hypotensive effect and antioxidant properties of Myrtus
communis L. growing in Algeria. Asian Pac. J. Trop. Biomed. 2015, 5, 19–28. [CrossRef]
46. Aidi Wannes, W.; Mhamdi, B.; Sriti, J.; Ben Jemia, M.; Ouchikh, O.; Hamdaoui, G.; Kchouk, M.E.; Marzouk, B. Antioxidant
activities of the essential oils and methanol extracts from myrtle (Myrtus communis var. italica L.) leaf, stem and flower. Food
Chem. Toxicol. 2010, 48, 1362–1370. [CrossRef]
47. Faisal, M.; Qahtan, A.A.; Alatar, A.A. Thidiazuron Induced In Vitro Plant Regeneration, Phenolic Contents, Antioxidant Potential,
GC-MS Profiles and Nuclear Genome Stability of Plectranthus amboinicus (Lour.) Spreng. Horticulturae 2023, 9, 277. [CrossRef]
48. Mokhtari, R.; Kazemi, F.M.; Rezaei, M.; Moftakharzadeh, S.A.; Mohseni, A. Antioxidant, Antimicrobial Activities, and Charac-
terization of Phenolic Compounds of Thyme (Thymus vulgaris L.), Sage (Salvia officinalis L.), and Thyme–Sage Mixture Extracts.
J. Food Qual. 2023, 2023, 2602454. [CrossRef]
49. Mackenzie, C.; Goerke, T.; Buecking, M.; Heidemann, M.; Leichtle, A.; Ringbeck, B.; Möllenkolk, F.; Ploch, M.; Bruchhage, K.-L.;
Pries, R. Determination of orally administered 1, 8-Cineol in nasal polyp tissues from chronic rhinosinusitis patients using gas
chromatography: Mass spectrometry. Sci. Rep. 2023, 13, 3605. [CrossRef] [PubMed]
50. Rodriguez-Amaya, D.B. Food carotenoids: Analysis, composition and alterations during storage and processing of foods. Forum
Nutr. 2003, 56, 35–37.
51. Machado-Carvalho, L.; Martins, T.; Aires, A.; Marques, G. Optimization of Phenolic Compounds Extraction and Antioxidant
Activity from Inonotus hispidus Using Ultrasound-Assisted Extraction Technology. Metabolites 2023, 13, 524. [CrossRef]
52. Nuzul, M.I.; Jong, V.Y.M.; Koo, L.F.; Chan, T.H.; Ang, C.H.; Idris, J.; Husen, R.; Wong, S.W. Effects of Extraction Methods on
Phenolic Content in the Young Bamboo Culm Extracts of Bambusa beecheyana Munro. Molecules 2022, 27, 2359. [CrossRef]
53. Alamri, F.B.; Sobahi, T.R.; Althagbi, H.I.; Abdel-Lateff, A.; Alfaifi, M.Y.; Mohammed, A.Y.; Abdel-Latif, E.; Alarif, W.M. Bioactivity
and molecular docking of lactones isolated from Centaurea pseudosinaica Czerep. Saudi Pharm. J. 2023, 31, 773–782. [CrossRef]
54. Munteanu, I.G.; Apetrei, C. Analytical Methods Used in Determining Antioxidant Activity: A Review. Int. J. Mol. Sci. 2021,
22, 3380. [CrossRef]
55. Amrita, K.I.; Sharma, A.D. Underutilized Plant Cymbopogan martinii Derived Essential Oil Is Excellent Source of Bioactives with
Diverse Biological Activities. Russ. Agric. Sci. 2023, 49, 100–117. [CrossRef]
56. Pinc, M.M.; Dalmagro, M.; Da Cruz, A.; Pereira, E.; Donadel, G.; Thomaz, R.T.; Da Silva, C.; Macruz, P.D.; Jacomassi, E.;
Gasparotto, J.A.; et al. Extraction Methods, Chemical Characterization, and In Vitro Biological Activities of Plinia cauliflora (Mart.)
Kausel Peels. Pharmaceuticals 2023, 16, 1173. [CrossRef]
57. Sacchetti, G.; Muzzoli, M.; Statti, G.A.; Conforti, F.; Bianchi, A.; Agrimonti, C.; Ballero, M.; Poli, F. Intra-specific biodiversity of
Italian myrtle (Myrtus communis) through chemical markers profile and biological activities of leaf methanolic extracts. Nat. Prod.
Res. 2007, 21, 167–179. [CrossRef]
58. Pourahmad, J.; Salimi, A.; Seydi, E. Role of oxygen free radicals in cancer development and treatment. In Free Radicals and Diseases;
IntechOpen: London, UK, 2016; p. 345.
59. Khan, M.; Sohail, R.N.I.; Asad, M.J.; Mashwani, Z.U. Antioxidant and hypoglycemic potential of phytogenic cerium oxide
nanoparticles. Sci Rep. 2023, 13, 4514. [CrossRef]
60. Diniz Do Nascimento, L.; Moraes, A.A.B.D.; Costa, K.S.D.; Pereira Galúcio, J.M.; Taube, P.S.; Costa, C.M.L.; Neves Cruz, J.; De
Aguiar Andrade, E.H.; Faria, L.J.G.D. Bioactive Natural Compounds and Antioxidant Activity of Essential Oils from Spice Plants:
New Findings and Potential Applications. Biomolecules 2020, 10, 988. [CrossRef]
61. Benchikh, F.; Amira, S.; Benabdallah, H. The evaluation of antioxidant capacity of different fractions of Myrtus communis L. leaves.
Annu. Res. Rev. Biol. 2018, 22, 1–14. [CrossRef]
62. Benchikh, F.; Benabdallah, H.; Amira, H.; Amira, I.; Mamache, W.; Amira, S. Free Radical Scavenging, Metal chelating and
Antiperoxidative Activities of M. communis Berries Methanol extract and its Fractions. Turk. J. Agric.-Food Sci. Technol. 2022, 10,
1089–1094. [CrossRef]
Molecules 2024, 29, 849 30 of 32
63. Kebsa, W.; Hassiba, R.; Mesbah, L. Quercetin protects liver cells and mitochondria against Doxorubicin induced oxidative stress
in Albinos’ rats. J. Biol. Act. Prod. Nat. 2015, 5, 331–338.
64. Iordache, A.M.; Nechita, C.; Podea, P.; S, uvar, N.S.; Mesaros., C.; Voica, C.; Bleiziffer, R.; Culea, M. Comparative Amino Acid
Profile and Antioxidant Activity in Sixteen Plant Extracts from Transylvania, Romania. Plants 2023, 12, 2183. [CrossRef]
65. Alhaithloul, H.A.; Alqahtani, M.M.; Abdein, M.A.; Ahmed, M.A.; Hesham, A.E.L.; Aljameeli, M.M.; Al Mozini, R.N.; Gharsan,
F.N.; Hussien, S.M.; El-Amier, Y.A. Rosemary and neem methanolic extract: Antioxidant, cytotoxic, and larvicidal activities
supported by chemical composition and molecular docking simulations. Frint. Plant Sci. 2023, 14, 1155698. [CrossRef] [PubMed]
66. Baeshen, N.A.; Almulaiky, Y.Q.; Afifi, M.; Al-Farga, A.; Ali, H.A.; Baeshen, N.N.; Abomughaid, M.M.; Abdelazim, A.M.; Baeshen,
M.N. GC-MS Analysis of Bioactive Compounds Extracted from Plant Rhazya stricta Using Various Solvents. Plants 2023, 12, 960.
[CrossRef]
67. Tawaha, K.; Alali, F.Q.; Gharaibeh, M.; Mohammad, M.; El-Elimat, T. Antioxidant activity and total phenolic content of selected
Jordanian plant species. Food Chem. 2007, 104, 1372–1378. [CrossRef]
68. Bawish, B.M.; Rabab, M.A.; Gohari, S.T.; Khattab, M.S.; Abdelkader, N.A.; Elsharkawy, S.H.; Ageez, A.M.; Zaki, M.M.; Kamel, S.;
Ismail, E.M. Promising effect of Geranium robertianum L. leaves and Aloe vera gel powder on Aspirin® -induced gastric ulcers
in Wistar rats: Anxiolytic behavioural effect, antioxidant activity, and protective pathways. Inflammopharmacology 2023, 31,
3183–3201. [CrossRef]
69. Kebsa, W.; Hassiba, R.; Mesbah, L. Polyphenolic fraction of Algerian propolis reverses doxorubicin induced oxidative stress in
liver cells and mitochondria. Pak. J. Pharm. Sci. 2014, 27, 1891–1897.
70. Chen, H.; Li, Y.; Wang, J.; Zheng, T.; Wu, C.; Cui, M.; Feng, Y.; Ye, H.; Dong, Z.; Dang, Y. Plant Polyphenols Attenuate DSS-Induced
Ulcerative Colitis in Mice via Antioxidation, Anti-Inflammation and Microbiota Regulation. Int. J. Mol. Sci. 2023, 24, 10828.
[CrossRef]
71. Alsahli, M.; Anwar, S.; Alzahrani, F.M.; Almatroudi, A.; Alfheeaid, H.; Khan, A.A.; Allemailem, K.S.; Almatroodi, S.A.; Rahmani,
A.H. Health Promoting Effect of Phyllanthus emblica and Azadiractha indica against Advanced Glycation End Products Formation.
Appl. Sci. 2021, 11, 8819. [CrossRef]
72. Karvekar, O.S.; Vadanagekar, A.S.; Sarvalkar, P.D.; Suryawanshi, S.S.; Jadhav, S.M.; Singhan, R.D.; Jadhav, J.P.; Sharma, K.K.K.;
Prasad, N.R. Bos taurus (A-2) urine assisted bioactive cobalt oxide anchored ZnO: A novel nanoscale approach. Sci. Rep. 2022,
12, 15584. [CrossRef]
73. Anwar, S.; Raut, R.; Alsahli, M.A.; Almatroudi, A.; Alfheeaid, H.; Alzahrani, F.M.; Khan, A.A.; Allemailem, K.S.; Almatroodi, S.A.;
Rahmani, A.H. Role of Ajwa Date Fruit Pulp and Seed in the Management of Diseases through In Vitro and In Silico Analysis.
Biology 2022, 11, 78. [CrossRef]
74. Mahdi, I.; Bakrim, W.B.; Bitchagno, G.T.M.; Annaz, H.; Mahmoud, M.F.; Sobeh, M.J. Unraveling the phytochemistry, traditional
uses, and biological and pharmacological activities of Thymus algeriensis Boiss. & Reut. Oxid. Med. Cell Longev. 2022, 25, 6487430.
75. Röhrl, J.; Piqué-Borràs, M.R.; Jaklin, M.; Werner, M.; Werz, O.; Josef, H.; Hölz, H.; Ammendola, A.; Künstle, G. Anti-Inflammatory
Activities of Arnica montana Planta Tota versus Flower Extracts: Analytical, In Vitro and In Vivo Mouse Paw Oedema Model
Studies. Plants 2023, 12, 1348. [CrossRef]
76. Babatuyi, C.Y.; Oyetayo, V.O.; Akinyosoye, F.A.; Oyeleye, I.S. Anti-inflammatory and analgesic potentials of fermented Citrullus
vulgaris with mutant and non-mutant strains of Bacillus subtilis to produce condiment (ogiri). Food Humanit. 2023, 1, 104–118.
[CrossRef]
77. Pham, T.V.; Ngo, H.P.T.; Nguyen, N.H.; Do, A.T.; Vu, T.Y.; Nguyen, M.H.; Do, B.H. The anti-inflammatory activity of the
compounds isolated from Dichroa febrifuga leaves. Saudi J. Biol. Sci. 2023, 30, 103606. [CrossRef] [PubMed]
78. Sharma, V.M.; Mathur, A.; Goyal, M.B.; Jat, S.L. The role of rectal diclofenac and aggressive hydration with Ringer’s lactate in
preventing post-endoscopic retrograde cholangiopancreatography pancreatitis in high-risk patients. Int. J. Gastrointest. Interv.
2023, 12, 87–92. [CrossRef]
79. Amira, S.; Dade, M.; Schinella, G.; Ríos, J.L. Anti-inflammatory, anti-oxidant, and apoptotic activities of four plant species used in
folk medicine in the Mediterranean basin. Pak J. Pharm. Sci. 2012, 25, 65–72. [PubMed]
80. Nassar, M.I.; Aboutabl El, S.A.; Ahmed, R.F.; El-Khrisy, E.D.; Ibrahim, K.M.; Sleem, A.A. Secondary metabolites and bioactivities
of Myrtus communis. Pharmacogn. Res 2010, 2, 325–329. [CrossRef]
81. Touaibia, M.; Chaouch, F. Anti-inflammatory effect of Myrtus nivellei Batt & Trab (Myrtaceae) methanolic extract. J. Funda. Appl.
Sci. 2015, 7, 77–82.
82. Morales, G.; Paredes, A.; Olivares, A.; Bravo, J. Acute oral toxicity and anti-inflammatory activity of hydroalcoholic extract from
Lampaya medicinalis Phil in rats. Biol. Res. 2014, 47, 6–13. [CrossRef]
83. Rossi, A.; Di Paola, R.; Mazzon, E.; Genovese, T.; Caminiti, R.; Bramanti, P.; Pergola, C.; Koeberle, A.; Werz, O.; Sautebin, L.
Myrtucommulone from Myrtus communis exhibits potent anti-inflammatory effectiveness in vivo. J. Pharmacol. Exp. Ther. 2009,
329, 76–86. [CrossRef]
84. Sumbul, S.; Ahmad, M.A.; Asif, M.; Akhtar, M. Myrtus communis Linn.—A review. Indian J. Nat. Prod. Resour. 2011, 2, 395–402.
85. Koeberle, A.; Pollastro, F.; Northoff, H.; Werz, O. Myrtucommulone, a natural acylphloroglucinol, inhibits microsomal
prostaglandin E2 synthase-1. Br. J. Pharmacol. 2009, 156, 952–961. [CrossRef]
86. Giampieri, F.; Cianciosi, D.; Forbes-Hernández, T.Y. Myrtle (Myrtus communis L.) berries, seeds, leaves, and essential oils: New
undiscovered sources of natural compounds with promising health benefits. Food Front. 2020, 1, 276–295. [CrossRef]
Molecules 2024, 29, 849 31 of 32
87. Al-Rajhi, A.M.H.; Qanash, H.; Almashjary, M.N.; Hazzazi, M.S.; Felemban, H.R.; Abdelghany, T.M. Anti-Helicobacter pylori,
Antioxidant, Antidiabetic, and Anti-Alzheimer’s Activities of Laurel Leaf Extract Treated by Moist Heat and Molecular Docking
of Its Flavonoid Constituent, Naringenin, against Acetylcholinesterase and Butyrylcholinesterase. Life 2023, 13, 1512. [CrossRef]
[PubMed]
88. Bhakta-Guha, D.; Guha, G. Unfurling the contradictory influence of PLA2 and its signaling mechanisms in determining the
antitumorigenic or protumorigenic fate of cells. Phospholipases Physiol. Pathol. 2023, 2, 31–43.
89. Mohapatra, S.; Prasad, A.; Haque, F.; Ray, S.; De, B.; Ray, S.S. In silico investigation of black tea components on α-amylase,
α-glucosidase and lipase. J. Appl. Pharm. Sci. 2015, 5, 42–47. [CrossRef]
90. Salentin, S.; Schreiber, S.; Haupt, V.J.; Adasme, M.F.; Schroeder, M. PLIP: Fully automated protein–ligand interaction profiler.
Nucleic Acid Res. 2015, 43, 443–447. [CrossRef] [PubMed]
91. Stojanović, S.; Zarić, S. Hydrogen bonds and hydrophobic interactions of porphyrins in porphyrin-containing proteins. Open
Struct. Biol. J. 2009, 3, 34–41. [CrossRef]
92. Omoboyowa, D.A.; Balogun, T.A.; Omomule, O.M.; Saibu, O.A. Identification of terpenoids from Abrus precatorius against
Parkinson’s disease proteins using in silico approach. Bioinform. Biol. Insight 2021, 15, 1–12. [CrossRef]
93. Omoboyowa, D.A.; Singh, G.; Fatoki, J.O.; Oyeneyin, O.E. Computational investigation of phytochemicals from Abrus precatorius
seeds as modulators of peroxisome proliferator-activated receptor gamma (PPARγ). J. Biomol. Struc. Dyn. 2023, 41, 5568–5582.
[CrossRef]
94. Omoboyowa, D.A. Sterols from Jatropha tanjorensis leaves exhibit anti-inflammatory potential: In vitro and in silico studies. Bull.
Natl. Res. Cent. 2021, 45, 1–13. [CrossRef]
95. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P. Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 2012, 64, 4–17. [CrossRef]
96. Yan, A.; Wang, Z.; Cai, Z. Prediction of human intestinal absorption by GA feature selection and support vector machine
regression. Int. J. Mol. Sci. 2008, 9, 1961–1976. [CrossRef] [PubMed]
97. Omoboyowa, D.A.; Omomule, O.M.; Balogun, T.A.; Saibu, O.A.; Metibemu, D.S. Protective potential of ethylacetate extract of
Abrus precatorius (Linn) seeds against HCl/EtOH-induced gastric ulcer via pro-inflammatory regulation: In vivo and in silico
study. Phytomedicine Plus 2021, 1, 100145. [CrossRef]
98. Compaore, S.; Belemnaba, L.; Koala, M.; Ouedraogo, N.; Thiombiano, A.; Ouedraogo, S. Consensus level in the traditional
management of diabetes and chemical potentiality of plants from north Sudanese, Burkina Faso. J. Med. Plants Res. 2020, 14,
415–427.
99. Dib, K.; Cherrah, Y.; Rida, S.; Filali-Maltouf, A.; Ennibi, O. In Vitro Antibacterial Activity of Myrtus communis L and Marrubium
vulgare L. Leaves against Aggregatibacter actinomycetemcomitans and Eikenella corrodens. Evid.-Based Complement. Altern. Med. 2021,
2021, 8351332. [CrossRef] [PubMed]
100. Siddiqui, N.; Rauf, A.; Latif, A.; Mahmood, Z. Spectrophotometric determination of the total phenolic content, spectral and
fluorescence study of the herbal Unani drug Gul-e-Zoofa (Nepeta bracteata Benth). J. Taibah Univ. Med. Sci. 2017, 12, 360–363.
[CrossRef]
101. Dewanto, V.; Wu, X.; Adom, K.K.; Liu, R.H. Thermal processing enhances the nutritional value of tomatoes by increasing total
antioxidant activity. J. Agric. Food Chem. 2002, 50, 3010–3014. [CrossRef]
102. Kardel, M.; Taube, F.; Schulz, H.; Schütze, W.; Gierus, M. Different approaches to evaluate tannin content and structure of selected
plant extracts—Review and new aspects. J. Appl. Bot. Food Qual. 2013, 86, 154–166.
103. Martin, H.; Baumann, B.; Andary, C.; Linsenmair, K.E.; McKey, D. Extraction and quantification of “condensed tannins” as a
measure of plant anti-herbivore defence? Revisiting an old problem. Naturwissenschaften 2002, 89, 519–524.
104. Cosmulescu, S.; Trandafir, I.; Nour, V. Phenolic acids and flavonoids profiles of extracts from edible wild fruits and their
antioxidant properties. Int. J. Food Prop. 2017, 12, 3124–3134. [CrossRef]
105. Khan, S.; Rehman, M.U.; Khan, M.Z.I.; Kousar, R.; Muhammad, K.; Haq, I.U.; Ijaz, K.M.; Almasoud, N.; Alomar, T.S.; Rauf, A.
In vitro and in vivo antioxidant therapeutic evaluation of phytochemicals from different parts of Dodonaea viscosa Jacq. Front.
Chem. 2023, 11, 1268949. [CrossRef]
106. Brands, S.; Schein, P.; Castro-Ochoa, K.F.; Galinski, E.A. Hydroxyl radical scavenging of the compatible solute ectoine generates
two N-acetimides. Arch. Biochem. Biophys. 2019, 15, 67. [CrossRef]
107. Dong, J.; Cai, L.; Xing, Y.; Yu, J.; Ding, Z. Re-evaluation of ABTS+ Assay for Total Antioxidant Capacity of Natural Products.
Natual Prod. Commun. 2015, 10, 2168–2172. [CrossRef]
108. Suseela, V.; Gopalakrishnan, V.K.; Varghese, S. In vitro antioxidant studies of fruits of Artemisia nilagirica (Clarke) Pamp. Indian J.
Pharm. Sci. 2010, 72, 644–649. [CrossRef]
109. Kar, B.; Kumar, R.S.; Karmakar, I.; Dola, N.; Bala, A.; Mazumder, U.K.; Hadar, P.K. Antioxidant and in vitro anti-inflammatory
activities of Mimusops elengi leaves. Asian Pac. J. Trop. Biomed. 2012, 2, 976–980. [CrossRef]
110. Karim, N.; Khan, I.; Khan, W.; Khan, I.; Khan, A.; Halim, S.A.; Khan, H.; Hussain, J.; Al-Harrasi, A. Anti-nociceptive and
Anti-inflammatory Activities of Asparacosin A Involve Selective Cyclooxygenase 2 and Inflammatory Cytokines Inhibition: An
in-vitro, in-vivo, and in-silico Approach. Front. Immunol. 2019, 10, 581. [CrossRef] [PubMed]
111. Kim, S.; Chen, J.; Cheng, T.; Gindulyte, A.; He, J.; He, S.; Li, Q.; Shoemaker, B.A.; Thiessen, P.A.; Yu, B.; et al. PubChem 2019
update: Improved access to chemical data. Nucleic Acid Res. 2019, 47, 1102–1109. [CrossRef] [PubMed]
Molecules 2024, 29, 849 32 of 32
112. Berman, H.M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.N.; Weissig, H.; Shindyalov, I.N.; Bourne, P.E. The protein data bank.
Nucleic Acid Res. 2000, 28, 235–242. [CrossRef] [PubMed]
113. He, M.M.; Smith, A.S.; Oslob, J.D.; Flanagan, W.M.; Braisted, A.C.; Whitty, A.; Cancilla, M.T.; Wang, J.; Lugovskoy, A.A.; Yoburn,
J.C. Small-molecule inhibition of TNF-α. Science 2005, 310, 1022–1025. [CrossRef]
114. Rondeau, J.M.; Ramage, P.; Zurini, M.; Gram, H. The molecular mode of action and species specificity of canakinumab, a human
monoclonal antibody neutralizing IL-1β. MAbs 2015, 7, 1151–1160. [CrossRef] [PubMed]
115. Trott, O.; Olson, A. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient
optimization, and multi-threading. J. Comput. Chem. 2010, 31, 455–461. [CrossRef] [PubMed]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.

Molecules 29 00849 v2

Uploaded by

Copyright:

Available Formats

Molecules 29 00849 v2

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Molecules 29 00849 v2

Uploaded by

Copyright:

Available Formats

molecules

Molecules 2024, 29, 849. https://doi.org/10.3390/molecules29040849 https://www.mdpi.com/journal/molecules

Polyphenols Flavonoids Condensed Hydrolysable

2.2. GC-MS Analysis

2.3. Antioxidant Activity

Molecules 2024, 29, x FOR PEER REVIEW 4 of 33

Figure 1. Representative GC-MS chromatogram of Myrtus communis L. methanolic extract. Peak

2.4. Anti-Inflammatory Activity

that of the well-known nonsteroidal anti-inflammatory drug diclofenac (DCF). A significant

Figure Figure 4. Inhibition

2.4.3. In Silico Study

Molecules 2024, 29, x FOR PEER REVIEW 12 of 33

Molecules 2024, 29, x FOR PEER REVIEW 13 of 33

Molecules 2024, 29, x FOR PEER REVIEW 14 of 33

Figure 10.10. Binding

Following visualization, hydrogen bonds, hydrophobic interactions, and pi-interactions

2.5. ADMET Results

Table 4. ADMET profiling of hit compounds in relation to diclofenac according to admetSAR.

3.2. Antioxidant Activity

3.3. Anti-Inflammatory Activity

3.3.2. In Vivo: Car-Induced Paw Edema

M. communis L. (50 mg.kg−1 ) effectively and significantly reduced inflammation through

3.3.3. In Silico Study

4. Materials and Methods

4.2. Collection of Plant Material

4.3. Methanolic Extract Preparation

weight of dry extract

4.4. Gas Chromatography–Mass Spectrometry (CG-MS) Analysis

4.5. Total Polyphenol Content (TPC)

4.6. Total Flavonoid Content (TFC)

4.7. Total Hydrolyzable and Condensed Tannin Content (THT, TCT)

where 5.2 × 10−2 is the cyanidine-equivalent constant, A is absorbance, V is the volume

4.8. Antioxidant Activity

vides outstanding potency information in pharmacological research by indicating how

4.8.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Scavenging Assay

4.8.2. H2 O2 Hydrogen Peroxide Scavenging Assay

4.8.3. Hydroxyl Radical (OH•) Scavenging Test

4.8.4. ABTS• Radical Scavenging Assay

4.8.5. Ferric-Reducing Antioxidant Power (FRAP)

4.9. Anti-Inflammatory Activity

4.9.2. In Vivo: Carrageenan-Induced Paw Edema in Rats

4.10. In Silico Pharmacokinetic Profile and Drug-Likeness Prediction

You might also like

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.