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Phytochemical Investigation, Antiulcer, Cyclooxygenase-2, and 15-

Lipoxygenase Inhibitory Activities of Echinops erinaceus Kit Tan

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DOI: 10.3390/separations10020076

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 separations
Article
Phytochemical Investigation, Antiulcer, Cyclooxygenase-2, and
15-Lipoxygenase Inhibitory Activities of Echinops erinaceus
Kit Tan
Sherouk Hussein Sweilam 1,2 , Fatma M. Abdel Bar 1,3 , Ahmed I. Foudah 1 , Mohammed H. Alqarni 1 ,
Omayma D. El-Gindi 2 , Moshera M. El-Sherei 4 and Essam Abdel-Sattar 4, *
Citation: Sweilam, S.H.; Abdel Bar,
F.M.; Foudah, A.I.; Alqarni, M.H.;
El-Gindi, O.D.; El-Sherei, M.M.;
Abdel-Sattar, E. Phytochemical
Investigation, Antiulcer,
Cyclooxygenase-2, and
15-Lipoxygenase Inhibitory Activities
of Echinops erinaceus Kit Tan.
Separations 2023, 10, 76. https://
doi.org/10.3390/separations10020076
Academic Editor: Paraskevas D.
Tzanavaras
Received: 18 December 2022
Revised: 8 January 2023
Accepted: 18 January 2023
Published: 21 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Separations 2023, 10, 76. https://doi.org/10.3390/separations10020076
1 Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University,
Al-Kharj 11942, Saudi Arabia
2 Department of Pharmacognosy, Faculty of Pharmacy, Egyptian Russian University, Cairo-Suez Road,
Badr City 11829, Egypt
3 Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
4 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562, Egypt
* Correspondence: essam.abdelsattar@pharma.cu.edu.eg
Abstract: Plants of the genus Echinop (Asteraceae) are traditional medicinal plants used to treat several
GIT ailments, owing to their diverse bioactive secondary metabolites, including sesquiterpenoids,
triterpenoids, phytosterols, phenolics, flavonoids, alkaloids, and essential oils. Echinops erinaceus
Kit Tan is a wild perennial herb of the genus Echinops which is endemic to Oman, Saudi Arabia,
and Yemen. Currently, there are no previous reports exploring its anti-ulcer and anti-inflammatory
effects. Additionally, few reports have described the chemical profile of E. erinaceus Kit Tan. In the
current study, the CHCl3 fraction of the aerial parts of the plant was subjected to chromatographic
isolation and spectroscopic identification via 1D and 2D NMR, and MS. The plant afforded two
new compounds, designated erinaceolic acid (E3) and erinaceoside (E5), in addition to five known
compounds, namely taraxasterol acetate (E1), taraxasterol (E2), apigenin (E4), stigmasterol-3-O-β-
D-glucoside (E6), and speranskoside (E7). The evaluation of the gastric ulcer protective activity
of the total extract and successive fractions of E. erinaceus, using the in vivo ethanol-induced ulcer
in rats model, revealed the significant effect of the tested extracts and fractions on the percentage
of gastric ulcer protection and ulcer index (500 mg/kg) compared to antodine (20 mg/kg). The
tested extracts and fractions also reduced the stomach contents of TNF-α and reduced IL-6 as
compared to the untreated group. Histopathological examination of the gastric mucosal tissues of
rats supportedprevious results. In addition, the main subfractions and their isolates were assessed for
their in vitro anti-inflammatory activity against COX-2 and 15-LOX enzymes. The new compounds
erinaceolic acid (E3) and speranskoside (E7) exhibited strong inhibition against COX-2 (3.41 and
2.62 µg/mL) and 15-LOX (10.05 and 5.51 µg/mL), respectively. A molecular docking study was
performed to reveal the binding interaction modes of the most active compounds against the binding
sites of COX-2 (PDB ID 3LN1) and 15-LOX (PDB ID 1LOX) proteins. Speranskoside (E7) showed a
dual binding affinity better than that of the cocrystallized references, celecoxib and (2E)-3-(2-oct-1-yn-
1-ylphenyl)acrylic acid (RS7) against both enzymes. This study shed a light on the potential use of
E. erinaceus in the protection and treatment of gastric ulcers.
Keywords: Echinops erinaceus; in vivo antiulcer; speranskoside; erinaceoside; erinaceolic acid; natural
COX-2 and 15-LOX inhibitors
1. Introduction
Inflammation is one of the compulsive pathological conditions that result from a
wide range of etiological aspects, such as diabetes, rheumatic, respiratory, chronic bowel,
immune-mediated, cardiovascular, and chronic kidney disorders. Peptic ulcers are one of
https://www.mdpi.com/journal/separations
Separations 2023, 10, 76 2 of 21

the most painful and bothersome inflammatory disorders and are mostly due to Helicobacter
pylori infection and the use of NSAIDs [1,2]. In folk medicine, peptic ulcers are treated
with several species from various plant families, mostly without scientific evidence. That
said, some clinical trials have proved that herbal medicines such as Aloe vera juice and the
rhizome of several Coptis spp. (syn. Coptidis rhizome) are effective treatments for gastric
ulcers [3,4].
Plants of the genus “Echinops” were used traditionally to treat GIT disorders such as
constipation [5], and have been shown to possess other related biological activities, such
as antiulcer, antidiarrheal, and spasmolytic activities [5–7], which may be attributed to
their bioactive classes, including alkaloids, phenolic compounds, phytosterols, terpenoids,
and essential oils [8]. The anti-inflammatory activities of the different extracts of several
Echinops species have been described in the literature [9–11]. In a previous study by our
research group, the preliminary screening of different extracts of the aerial parts of the
Saudi wild medicinal plant E. erinaceus against COX-1, COX-2, and 15-LOX enzymes has
been described. The ethyl acetate (EtOAc) and chloroform (CHCl3 ) fractions exhibited
promising inhibitory activity against 15-LOX (IC50 2.2 µm and 2.9 µm, respectively) [12].
Additionally, chemical and biological investigations of the CHCl3 extract of the same
plant led to the isolation of diverse phytochemical classes, including unsaturated fatty
acid esters (such as methyl and ethyl oleate), pseudoguaiane sesquiterpenes (such as
erinaceosin), benzofurans (such as loliolide), phenolics (such as E-p-coumaric acid and
5,7,3‘,5‘-tetrahydroxy flavanone), and abscisic alcohol derivatives (such as erinaceol) [13].
Biological evaluations of the extracts and/ or bioactive compounds from this plant showed
antioxidant, cytotoxic, and antimicrobial activities [13]. Additionally, the anti-inflammatory
activity of taraxasterol acetate isolated from E. echinatus Roxb. and E. spinosus was reported
against carrageenan and formaldehyde-induced inflammations in animal models [14].
Likewise, apigenin isolated from the whole E. echinatus Roxb. showed in vitro antifungal
effects against the conidia of Alternaria tenuissima (Kunz. ex Pers.) [14]. The sesquiterpene
glycoside, macrochaetoside A and B, and the triterpenoid cyclostenol from E. macrochaetus
Boiss. showed cytotoxic activity towards three cancer cell lines, including MCF-7, HEPG-2,
and HCT-116 [15]. The hexane extract of the E. spinosissimus Turra subsp. Spinosus was
shown to accumulate β-sitosterol (44.97%) and stigmasterol (34.95%) of the total phytosterol
content, and was demonstrated to have antibacterial activity against a panel of Gram-
positive and Gram-negative bacteria using the diffusion disc and broth micro-dilution
assays [16].
Therefore, the present study aimed to isolate and structurally characterize further phy-
tochemicals from the bioactive CHCl3 fraction of E. erinaceus. Moreover, in vivo assessment
of the protective effect of the total methanol extract and successive fractions of E. erinaceus
against ethanol-induced gastric ulcers in rats, including estimation of the gastric contents
of TNF-α and IL-6, was carried out. Additionally, in vitro evaluation of the inhibitory
activity of the main bioactive CHCl3 fractions and their isolated compounds against the
pro-inflammatory enzymes, COX-2 and 15-LOX, using in vitro and in silico methods, was
performed. Furthermore, in silico prediction of the pharmacokinetics and drug-likeness
properties of the investigated compounds were discussed.

2. Materials and Methods

2.1. Plant Material
The aerial parts of Echinops erinaceus Kit Tan were collected from the local desert in
Riyadh, Saudi Arabia in March 2018. The plant was identified as mentioned before [12,13].
The powdered plant was extracted and fractionated according to the method reported
previously [12,13].

2.2. General Experimental Procedures

The solvents of analytical grade and sephadex LH-20 were purchased from Sigma-
Aldrich (St. Louis, MO, USA). Silica gel 60 type A (70–230 mesh), and RP-Silica gel
Separations 2023, 10, 76 3 of 21

(25–40 µm) were obtained from Merck (Darmstadt, Germany). The pump of MPLC RHSY
Synchronous Metering Pump was purchased from Fluid Metering, Inc (Palo Alto, CA,
USA). TLC plates were visualized by spraying with a 10% methanolic solution of vanillin-
sulfuric acid (H2 SO4 ), followed by heating to 100 ◦ C. The 1D and 2D NMR were obtained
on a Bruker UltraShield Plus 500 MHz spectrometer (Bruker, Fällanden, Switzerland)
at 500 MHz for 1 H and 125 MHz for 13 C-NMR, while HRESIMS spectra were acquired
on a UPLC RS Ultimate 3000-Q in negative- or positive-ion mode. Optical activity was
measured on a Perkin-Elmer Model 341 LC polarimeter (PerkinElmer, Waltham, MA, USA)
at ambient temperature.

2.3. Isolation and Purification of Compounds from the CHCl3 Fraction

The CHCl3 (40 g) fraction was subjected to column chromatography (CC) over silica gel
(850 g). Elution was achieved with gradient elution, using a mixture of CHCl3 -MeOH with
increasing polarity from 100:0 to 0:100 v/v, and fractions of 150 mL were collected. Similar
fractions were combined together based on TLC monitoring to give a total of fourteen
major fractions. Fraction-1 (1.2 g) was purified by chromatography on Si gel columns
using a mixture of ethyl acetate/n-hexane (0:100 to 10:90) followed by an RP-18 column
(iso-propanol-water, (6–4 till 10–0), v/v) to yield compound E1 (53 mg). Fraction-2 (0.2 g)
was treated similarly to Fraction-1 to yield compound E2 (53 mg). The Fraction-3 (1.0 g)
was subjected to Si gel column chromatography eluted with n-hexane-ethyl acetate (1:9 →
1:1, v/v) to give nine subfractions (Fractions-3 I-IX). Subfraction Fraction-3-III was purified
by re-chromatography on a Si RP-18 column (MeOH-H2 O, 3:7, v/v) to give compound
E3 (15 mg). The Fraction-4 (1.0 g) was chromatographed using a Si gel RP-18 column
(methanol-water, 3:7 to 6:4, v/v) to give six main fractions (Fractions-4-I-VI). Fraction-4-III
(300 mg) afforded compound E4 (40 mg) and compound E5 (18 mg) by purification on a
sephadex LH-20 column, and on RP-18 using MeOH-H2 O (4–6 to 10–0, v/v). Fraction-5
(1.5 g) was subjected to chromatography on Si gel column eluted with EtOAc/n-hexane
(10–100%) followed by MeOH/EtOAc (10–100%) to yield six subfractions (Fractions-5-I-
VI). Fraction-5-VI (700 mg) was purified on a Si gel column using 5% MeOH/CHCl3 and
afforded compound E6 (100 mg). Fraction-6 (2.2 g) was purified on a sephadex LH-20
column to yield compound E7 (20 mg). The isolation procedure of compounds is outlined
in flowcharts S1–S3.

2.4. In Vivo Anti-Ulcer Assay

2.4.1. Animals
Acute Toxicity Assay
Twenty Swiss mice (20–30 g) of both sexes were fasted overnight and provided with
water. Mice were randomly divided into four groups of five animals each. The crude
methanol extract was administered via oral gavage. The procedure was repeated for further
higher doses of 10, 50, 200, and 500 mg/kg, and the mice were observed for any mortality
for 48 h.

Anti-Ulcer Assay
Forty Sprague Dawley male rats (140–150 g) for the anti-ulcer assay and Swiss mice
(20–30 g) both sex for the acute toxicity assay were provided by the animal house of
the National Research Centre (Cairo, Egypt). The animal experiments were performed
according to the laboratory guidance of the recommendations of the health Guide for the
Care and Use of Laboratory Chemicals and Animals with ethical approval number: MP
(2255) and MP (3086).

2.4.2. Induction of Gastric Ulcer and Preparation of Tissue Homogenate

Anti-ulcer activity was evaluated in an ethanol-induced gastric ulcer model in rats
according to the method described before [17]. The animals were fasted for 24 h with free
access to water. Rats were randomly assorted into eight groups of five rats. Group I (control
Separations 2023, 10, 76 4 of 21

gp) was composed of normal rats that received saline vehicle (1ml/kg). Rats in Group II
(ethanol control) received a single intragastric dose of absolute ethanol (1 mg/kg). Group
III received a single intragastric dose of ethanol + antodine (20 mg/kg, Sigma-Aldrich, St.
Louis, MO, USA). Groups IV-VIII received a single intragastric dose of ethanol + a single
intragastric dose (500 mg/ kg) of the five crude extracts of E. erinaceus separately. Rats were
given treatments and antodine 1 hr before ulcer induction by oral gavage.
The stomach was placed in ice-cold phosphate buffer (pH 7.4) to prepare the 20%
homogenate using a tissue homogenizer (MPW−120, Bit-Lab Medical instruments, Poland).
Homogenized tissues were centrifuged at 4000 rpm/min for 10 min at 4o C using a cooling
centrifuge (Laboratory Centrifuge, 2K15, B. Braun Sigma Co., Melsungen, Germany). The
supernatant was collected and stored at −80 ◦ C and then used for estimation of the gastric
contents of tumor necrosis factor-α, TNF-α, and interleukin-6, IL-6 (SinoGeneClon Biotech
Co., Ltd., Hangzhou, China).

2.4.3. Determination of Gastric Ulcer Index (UI) and Percentage of Inhibition

At the end of the experiment, the animals were euthanized under deep ether anesthesia
1 h post ethanol instillation. Following immediate laparotomy, the stomachs were excised,
opened along the greater curvature, and rinsed with normal saline to remove gastric
contents and blood clots. Stomachs were blotted dry and macroscopically inspected for
gross gastric injury (expressed as ulcer index) [18]. The overall total diameter of ulcers
in one stomach divided by factor 10 was designated as the ulcer index (UI) [19]. The
percentage of protection was calculated by the following formula:

[(UI ethanol control—UI treated)/UI ethanol control] × 100

2.4.4. Determination of Gastric Content of TNF-α and IL-6

The gastric contents of TNF-α and IL-6 were determined using ELISA (Enzyme-Linked
Immunosorbent Assay) kit and the results were calculated by following the manufacturer’s
instructions (NOVA kit, Beijing, China). Standards and samples were pipetted into wells
with immobilized antibodies specific for rat TNF-α and IL-6 and then incubated. TMB
(tetramethylbenzidine) substrate solution was added to the wells; color developed propor-
tionally to the amount of TNF-α and IL-6 bound. Color development was then discontinued
(using Stop Solution) and its intensity was measured at 450 nm.

2.5. In Vitro COX-2 and 15-LOX Enzyme Assay

The in vitro ability of the test compounds to inhibit the COX-2 enzyme was determined
using a COX-2 inhibitor screening assay kit (catalog number k547, Biovision, Waltham, MA,
USA), and their ability to inhibit 15-LOX enzymes (IC50 values, µg/mL) was determined
using a human recombinant enzyme assay kit (catalog no 760700, Cayman Chemical, Ann
Arbor, MI, USA) according to the manufacturer’s instructions. The test samples were
dissolved in DMSO and tested at concentrations ranging from 500 to 0.25 µg/mL in a final
volume of 1 mL, or in the vehicle (DMSO, 1.0%). Stock solutions were freshly prepared
before use, and buffer solution (0.1M Tris HCl, pH, 7.4) was used. Nordihydroguairetic
acid (NDGA) was used as a positive control for the COX-2/15-LOX inhibition assays.
The samples were tested at twelve different concentrations (0.25, 0.5, 1, 2, 3.9, 7.8, 15.6,
31.25, 62.5, 125, 250, 500 µg/mL) in a final volume of 210 Ml in a triplicate manner. The
concentration of the test compound which caused 50% inhibition (IC50 ) was calculated
from the concentration–response curve using GraphPad Prism software, version 5 (San
Diego, CA. USA). The 15-LOX was determined by measuring the increase in absorbance at
490 nm using a Microplate reader (Biotek, Winooski, VT, USA) [20,21].
Separations 2023, 10, 76 5 of 21

2.6. Docking Study

2.6.1. Protein-Ligand Docking
Marvinsketch (https://chemaxon.com/ accessed on 27 June 2022), ADME prediction
web tool (http://www.swissadme.ch/ accessed on 27 June 2022), PASS prediction web tool
(http://www.way2drug.com/PASSOnline/index.php accessed on 25 April 2022). PyRx
software, via AutoDock Vina 4.2 version and Discovery Studio Visualizer software, 2021
version, were used to generate the scoring functions (binding affinities) and visualization of
the protein–ligand interactions involving non-bonding polar and hydrophobic interactions
was accomplished using ChimeraX 1.3 software version. The ligand–protein interactions
were investigated using COX-2 (PDB ID: 3LN1) [22] and 15-LOX (PDB ID: 1LOX) pro-
teins [23]. The calculated binding free energies (E) and the binding interactions within the
active site of different studied targets were determined.

2.6.2. Molecular Docking Analysis

The molecular docking analysis was performed as previously described [23,24]. The
conformation of the molecule ligands within the appropriate target binding site of COX-2
(PDB: 3LN1) and 15-LOX (PDB: 1LOX) was accomplished using PyRx virtual screening
tool software and Autodock 4 and Autodock Vina (The Scripps Research Institute, La Jolla,
CA, USA) and Pymol v2.5.2 (Schrodinger, New York, NY, USA). Discovery Studio 2021
(Dassault Systemes, Vélizy-Villacoublay, France) was used to determine the manner of the
contact and to visualize it in two dimensions, while UCSF ChimeraX 1.3 (San Francisco, CA,
USA) was utilized to depict the molecules and interaction residues in three dimensions.

2.7. Statistical Analysis

The results were expressed as the mean ± S.D, and statistical comparisons were
carried out using one-way analysis of variance (ANOVA) followed by Tukey’s multiple
comparisons test. GraphPad Prism software, version 5 (Graph Pad Inc., San Diego, CA,
USA) was used for all statistical tests. The difference was considered significant when
p < 0.05 for the anti-ulcer assay.

3. Results and Discussion

3.1. Identification of the Isolated Compounds
3.1.1. Identification of Compounds E1 and E2
The 1 H- and 13 C-NMR spectra of E1 (Table S1, Figures S1 and S2) suggested a penta-
cyclic triterpenoid which was confirmed as taraxasterol acetate by comparison with the
published data [25]. The 1 H- and 13 C-NMR spectra of compound E2 (Table S1, Figures
S3 and S4) had a great similarity to that of E1. The major differences between the two
compounds were the absence of the acyl group in E1 (δH 1.98, δC 21.8 and 171.5) and
the upfield shift of the oxymethine signal of C-3 (δH 3.19, s; δC 79.0) in E2. Thus, E2 was
identified as taraxasterol [25].

3.1.2. Identification of Compound E3

Compound E3 was isolated as colorless needles (m.p. 230–233 ◦ C; [α]25 D – 23.3,
c -0.02, CH3 OH). Compound E3 was analysed and identified from the 1D, 2 DNMR and MS
spectral data (Table 1, Figures S5–S12). The molecular formula was deduced as C14 H22 O5
based on the [M]+ ion at m/z 270.1467 [M+ ] (calcd. 270.3250) in HRESIMS (Table S2, Figure
S12). The 1 H-NMR, APT, DEPT, and HSQC spectra data of E3 (Table 1, Figures S5–S9)
showed the presence of fourteen carbon signals. The presence of a cyclopentane ring
system was evident from the presence of five aliphatic carbon signals distinguished into
the following: an oxygenated carbon at δC 71.4 (C-1), a methine carbon signal at δC 64.9
(δH 3.68, C-3), two methylenes at δC 41.2 (δH 2.23 and 1.58, C-2) and 48.0 (δH 1.51 and
1.21, C-4), and a quaternary aliphatic carbon at δC 36.6 (C-5). The 1 H-NMR spectrum
of E3 displayed two trans olefinic protons at δH 7.11 (d, J = 15.8 Hz, H-6) and 6.10 (d,
J = 15.8 Hz, H-7) of an α, β-unsaturated carbonyl residue. It also showed a downfield
Separations 2023, 10, 76 6 of 21

methyl singlet at δH 2.22 (δC 28.0, H-9), which was correlated in the HMBC spectrum
(Figure S10) with a ketonic group at δC 201.0 (C-8) and with the olefinic carbons at δC 146.2
(C-6) and 134.3 (C-7). Moreover, the HMBC correlations of the proton signal at δH 6.10
(H-7) with the ketonic group (C-8)and with the adjacent carbons—including δC 146.2 (C-6),
28.0 (C-9), and 71.4 (C-1)—confirmed the presence of a 3-oxobut-1-en-1-yl side chain at
C-1 of the cyclopentane ring, similar to the side chain in the closely related compound
4-hydroxy-3,5,5-trimethyl-4-[(1E)-3-oxobut-1-en-1-yl] cyclohex-2-en-1-one [26].

Table 1. NMR spectral data for the compound E3 in CD3 OD.

HMBC (H→C) ab COSY a

C/H No. Type 1 H-NMR a 13 C-NMR b
2J 3J 4J 1 H-1 H
CH CH CH

1 C 71.4
2.23, q (5.1) c
2 CH2 41.2 C-1, C-3 C-4, C-6, C-10 C-7 H-3
1.58, d (9.2, 5.1)
3 CH 3.68, m 64.9 C-4 C-5 H-2, H-4
1.51, d (10.7); C-3, C-1, C-2,
4 CH2 48.0 H-3
1.21, d (12.8) C-5 C-13/14
5 C 36.6
C-1,
6 CH 7.11, d (15.8) 146.2 C-8 C-9
C-7
C-6, C-1,
7 CH 6.10, d (15.8) 134.3
C-8 C-9
8 C=O 201.0
9 CH3 2.22, s 28.0
10 C 69.4
11 C=O 178.5
12 CH3 1.10, s 20.6 C-10 C-3
13 CH3 1.11, s 30.3 C-14 C-5
14 CH3 0.90, s 26.6 C-1, C-4, C-13
a b c
Measured at 500 MHz, measured at 125 MHz, overlapped signal.

The previous spectral data (Table 1, Figures S5–S12) confirmed the presence of an
undescribed cyclopentane ring system attached to a side chain. Further connectivity of
the cyclopentane ring system with the side chain was confirmed from the very long-range
or non-standard HMBC correlations (n JH-C n > 3) (Figure 1 and Figure S10) [27]. The
presence of a 2-hydroxy propionic side chain at C-3 was confirmed by comparison with
the side chain in 2-hydroxy-2-(p-tolyl)propanoic acid [28]. The relative configuration of
E3 was determined by analysis of its NOESY spectrum and from the determination of the
coupling constants (Figure S11). In particular, the NOESY correlations of δHH H-7/H-2
and H-14/H-4 indicated their α-configuration, and those between H-6/H-13, H-6/H-2,
H-12/H-3, H-12/H-4, H-13/H-2, and H-13/H-12 indicated their β-configuration. Finally,
the structure of compound E3 was established and confirmed by mass fragmentation as
E-2-hydroxy-2-(3-hydroxy-4,4-dimethyl-3-(3-oxobut-1-en-1-yl) cyclopentyl)-propanoic acid
and was named erinaceolic acid. To the best of our knowledge, this is the first report of this
compound from nature.
Separations 2023,
Separations 10,10,
2023, 76 76 7 of2221
7 of

Figure1.1.Chemical
Figure Chemicalstructures
structures ofof compounds
compounds E1–E7,
E1–E7, HMBC
HMBC correlations
correlationsofofE3E3and
andE5,
E5,and
andmass
mass
fragmentation pattern of E3: Taraxasterol acetate (E1), Taraxasterol (E2), erinaceolic acid (E3), apig‐
fragmentation pattern of E3: Taraxasterol acetate (E1), Taraxasterol (E2), erinaceolic acid (E3), apigenin
enin (E4), erinaceoside (E5), stigmasterol‐3‐O‐β‐D‐glucoside (E6), and naringenin‐7‐O‐β‐D‐(4″‐trans‐
(E4), erinaceoside (E5), stigmasterol-3-O-β-D-glucoside (E6), and naringenin-7-O-β-D-(4”-trans-p-
p‐coumaroyl) glucoside (E7).
coumaroyl) glucoside (E7).
Table 1. NMR spectral data for the compound E3 in CD3OD.
3.1.3. Identification of Compound E4
Compound E4 was1 identified 13C‐
as apigenin HMBC its
by comparing 1 H-NMR
(H→C) ab
and APT COSY a
spectra
C/H No. Type H‐NMR a
with a similar reported structure (Figures NMRS13 and
b 2J CH
S14) [29]. 3J CH 4J CH 1H‐1H

1 C 71.4
3.1.4. Identification of 2.23,
Compound
q (5.1) c E5
2 CH2 41.2 C‐1, C‐3 C‐4, C‐6, C‐10 25 C‐7 H‐3
Compound E5 1.58, (9.2, 5.1)as yellow amorphous powder ([α]D – 235.5, c. 0.24,
was disolated
CH3 OH).3 Compound
CH was identified
E5 3.68, m from the detailed
64.9 C‐4 studyC‐5of its 1D, 2 DNMR H‐2,and
H‐4MS
spectral data (Table 2, 1.51,
Figures S15–S21).
d (10.7); The molecular
C‐3, formula was
C‐1, C‐2, deduced as C 26 42 O8
H
4 CH 2 +
based on the [M+H] 1.21, ion at 48.0
m/z 483.2975 [M+H] + H‐3
d (12.8) C‐5 (calcd. C‐13/14
483.2913) in HRESIMS (Table
S3, Figure S21). From the 1 H- and 13 C-NMR spectral data, the result of acid hydrolysis
5 C 36.6
and positive test for sugar and/or glycoside revealed the presence of one sugar moiety,
1 H-, 13146.2
C‐1,
6 as β-D-glucose.
identified CH 7.11, dThe
(15.8) C-NMR, APT, DEPT, and C‐8 HMBC C‐9 spectra (Table 2,
C‐7
Figures S15–S20) of E5 revealed the presence of twenty six carbon signals ascribed to the
C‐6, C‐1,
presence7 of a basic
CH skeleton
6.10, dof(15.8)
a pseudoguaiane
134.3 sesquiterpene [30], one glucose moiety and
C‐8 C‐9
an isovaleroyl moiety. However, the presence of an α,β-unsaturated cyclopentenone ring
8 C=O 201.0
in the pseudoguaiane skeleton was suggested based on the presence of a ketonic group at
δC 202.59 (C-3),CH 2.22, olefinic
an3 endocyclic s 28.0 at δ 125.9 (C-2, δ 5.76 s), and a quaternary
bond C H
10 C 69.4
carbon signal at δC 180.2 (C-1), and was confirmed by the 3 J- and 4 J- HMBC correlations of
11 C-4,C=O
H-2 with C-5, and C-9. The presence 178.5of two methyl singlets at δH 1.14 (δC 23.3, H-12)
12 (δC 25.3,
and 1.16 CH3H-13), which
1.10, s were correlated
20.6 C‐10
in C‐3
the HMBC spectrum to the oxygenated
quaternary
13 carbon
CH3 at δC 80.9
1.11,(C-11)
s and30.3
the methine
C‐14 carbon atC‐5 δC 42.6 (C-7), indicated the
presence
14 of a hydroxy-isopropyl
CH3 0.90, s group attached
26.6 to C-7. The presence
C‐1, C‐4, C‐13of a β-glucose moiety
was evident from the proton signal at δH 4.45 (d, J= 7.4 Hz) which was correlated in the
HSQC spectrum with anomeric carbon at δC 99.1 (C-1‘). This was further confirmed by
Separations 2023, 10, 76 8 of 21

the presence of four oxygenated methines at δC 75.3 (C-3‘), 75.7 (C-2‘), 78.5 (C-5‘), and 72.5
(C-4‘), and a methylene at δC 65.4 (C-6‘). The downfield chemical shift of C-11 (δC 80.9)
and the HMBC correlation between C-11 and to the anomeric proton (Table 2, Figure S20)
confirmed the position of the glycosylation site at C-11. An isovalerate moiety was evident
from the existence of five proton/carbon signals, including two methyl doublets at 0.91
(6H, J= 6.4 Hz, δC 23.3, H-4“/ H-5“), a methylene at δH 2.12 (2H, d, J = 6.6 Hz, δC 44.6,
H-2“), a methine at δH 2.01 (m), δC 27.2, H-3“, and finally an ester carbonyl group at δC
174.8 (C-1“) [15]. The acylation position at C-6‘ was evident from the downfield shift of C-6
(δC 65.4) and further confirmed by the 3 J-HMBC correlation of the H2 -6‘ of the sugar moiety
(δH 3.97 and 4.42) with C-1“ (δC 174.8). From the aforementioned findings, the structure of
E5 was confirmed as ∆1(2) , 3-oxo-pseudoguaien-11-β-D-glucopyranosyl-6‘-isovalerate or
ambrosanoli-1(2)-en-3-oxo-11-O-[6‘-O-isovaleryl-β-D-glucopyranoside], which was named
erinaceoside, a new natural compound.

Table 2. NMR spectral data for the compound E5 in CD3 OD.

13 C-NMR b
HMBC (H→C) a/b COSY a
C/H No. Type 1 H-NMR a
2J 3J 4J 1 H-1 H
CH CH CH
Aglycone moiety
1 C 180.2
2 CH 5.76, s 125.9 C-4, C-5 C-9, C-15
3 C=O 202.5
4 CH2 2.22, 2.28, m 43.5 C-3, C-5 C-15 C-10
5 C 41.9

CH2 35.2 C-5, C-7 C-8, C-11, C-10, C-12 H-7

6 1.25, brd (12.9); 2.02, m C-15
7 CH 1.67, m 42.6 C-11 H-6, H-8
8 CH2 1.67, m 27.5 C-7 H-7, H-9
9 CH2 2.20, m; 2.51, m 30.6 C-10 C-5 H-10, H-8
10 CH 2.29, m 36.0 C-14 C-5 C-15 H-14
11 C 80.9
12 CH3 1.14, s 23.3 C-11 C-7, C-13 C-6, C-1‘
13 CH3 1.16, s 25.3 C-11 C-7, C-12 C-6, C-1‘
14 CH3 0.95, d (5.0) 16.3 C-10 C-3 H-10
15 CH3 1.02, s 20.1 C-5 C-6 C-10
Glucose moiety
1‘ CH 4.43, d (5.4) 99.1 C-2‘ C-3‘, C-11 C-4‘ H-2‘
2‘ CH 3.10, m 75.7 C-1‘ C-5‘ H-1‘, H-3‘
3‘ CH 3.39, m 75.3 C-6‘ H-2‘, H-4‘
4‘ CH 3.16, m 72.5 C-3‘, C-5‘ C-6‘ C-1‘ H-5‘
5‘ CH 3.33, m 78.5 C-4‘ C-3‘ C-2‘ H-4‘, H-6‘

6‘ CH2 3.97, dd (7.25, 11.7); 65.4 C-2‘ C-11, C-3‘ C-4‘ H-2‘
4.42, d (7.4)
Isovaleric acid moiety
1“ C 174.8
2“ CH2 2.12, d (6.6) 44.6 C-3“ C-4“, C-5“ H-3“

3“ CH 2.01, m 27.2 C-2“, C-4“, C-1“ H-2“, H-4“,

C-5“ H-5“
4“ CH3 0.91, d (6.4) 23.3 C-3“ C-2“, C-5“ H-3“
5“ CH3 0.91, d (6.4) 23.3 C-3“ C-2“, C-4“ H-3“
a b
Measured at 500 MHz, Measured at 125 MHz.
Separations 2023, 10, 76 9 of 21

3.1.5. Identification of Compound E6

Comparison of the spectral data of E6, including 1 H-NMR, APT, COSY, HSQC, and
HMBC (Table S4 and Figures S22–S26) with those published in the literature [31–33] con-
firmed this compound’s assignment as stigmasterol-3-O-β-D-glucoside. It is worth noting
that compounds E-1, E-2, E4 and E6 were identified for the first time from E. erincaeus
(Figure 1).

3.1.6. Identification of Compound E7

Compound E7 was obtained as a buff solid (m.p. 253 ◦ C). It had a molecular formula
of C30 H28 O12 deduced from the 13 C-NMR, APT, and DEPT spectra and from the pseudo-
molecular ion peak at m/z 603.1423 [M+Na]+ (calcd. 603.1478) in positive HRESIMS mode,
and 579.1503 [M-H]− (calcd. 579.1502), and 615.1271 [M+ Cl]− (calcd. 615.1269) in negative
HRESIMS mode. The result of acid hydrolysis and the spectral data (1 H-, 13 C-NMR, APT,
and DEPT) of E7 (Table 3, Figures S27–S33) revealed the presence of one sugar moiety, a
phenolic, and a flavanone skeleton identified as β-D-glucose, trans-p-coumaric acid and
naringenin. The flavanone part was identified as naringenin, based on the 1 H-NMR spec-
trum (Table 3, Figure S27) which showed the presence of six aromatic protons, including
two meta-coupled protons at δH 6.18 (d, J= 2.5 Hz, H-6) and 6.20 (d, J = 2.2 Hz, H-8) of the
A-ring of the flavanone moiety, two pairs of doublet protons of para-substituted aromatic
rings at δH 7.34 (d, J= 8.1, 2H, H-2‘/H-6‘), and 6.80 (d, J= 8.2 Hz, 2H, H-3‘/H-5‘) of the B-ring.
In addition, three signals resonating at δH 5.50 (dd, Jax . = 12.6, 2.05 Hz), 3.42 (m), and 2.75
(br.d, Jeq .= 17.0 Hz) were assigned to H-2, H-3ax , and HB-3eq . protons, respectively, of the
C-ring [29,34]. The presence of a 7-O-β-glucosyl substitution was evident from the HMBC
correlation between the proton signal at δH 5.13 (H-1“) and that at C-7 (δC 165.3). The
phenolic acid moiety was identified as trans-p-coumaric acid from the pair of ortho-coupled
protons at δH 7.57 (d, J= 8.57, Hz, 2H, H-2“‘/H-6“‘) and 6.80 (d, J= 8.2 Hz, 2H, H-3“‘/H-5“‘,
H-7“‘) of the para-substituted benzene ring observed, and from the carbon signals reso-
nances at δc 166.1 (C-9“‘), 160.1 (C-4“‘), 145.3 (C-7“‘), 130.6 (C-2“‘/C-6“‘), 125.3 (C-1“‘), 115.4
(C-3“‘/C-5“‘), and 114.3 (C-8“‘) [35]. The presence of a coumaric acid moiety was further
confirmed by the presence of a characteristic fragment at m/z 273.0734 [M—coumaroyl
glucose + 2H] (calcd. 273.0763), coincident with C14 H19 O5 • (Table S5, Figures S32 and S33).
Thus, the structure of E7 was confirmed as naringenin-7-O-β-D-(4“-trans-p-coumaroyl) glu-
coside (speranskoside), isolated for the first time from the Asteraceae family, but reported
before from Speranskia tuberculata (Euphorbiaceae) [36,37].
Separations 2023, 10, 76 10 of 21

Table 3. NMR spectral data of compound E7 in DMSO-d6 .

HMBC (H→C) a/b COSY a HMBC (H→C) a/b COSY a

C/H No. Type 1
H-NMR a 13
C-NMRb C/H No. Type 1
H-NMR a 13
C-NMRb
2 3 4 1 1 2 3 4 1
JCH JCH JCH H- H JCH JCH JCH H-1 H
Aglycone moiety (DMSO-d6 ) Glucose moiety (DMSO-d6 ,)
2 CH 5.50, dd (12.6, 2.05 Hz) 78.9 C-2‘/6‘ H-3 1“ CH 5.13, d, J= 7.8 Hz 99.5 C-7 H-2“
3 ax. 3.42, m c C-2, 4 C-6‘ H-2 2“ CH 3. 37, br.s c 73.9 H-1“
CH2 42.3
3 eq. 2.75, brd (17.0 Hz) C-4 H-2 3“ CH 3.57, br.s c 73.3
4 C 197.9 4“ CH 4.76, br.s 70.9 C-5“, 6“ C-9“‘
5 C 12.08, s 163.0 5“ CH 3.75, br.s 74.9
6 CH 6.18, d (2.5 Hz) 96.7 C-5 C-8 6“ CH2 3.37, br.s c 60.6
7 C 165.3 Trans-p-coumaroyl moiety (DMSO-d6 )
8 CH 6.2, d (2.2 Hz) 95.7 1“‘ C. 125.3
9 C 158.0 2“‘ CH 7.57, d (8.57 Hz) 130.6 C-1“‘, 3“‘ C-4“‘, C-5“‘, H-3“‘
6“‘, 8“‘
7“‘
10 C 103.6 3“‘ CH 6.80, d (8.2 Hz) 115.4 C-4“‘ C-1“‘ H-2“‘
1‘ C 125.2 4“‘ C-OH 12.08, s 160.1
2‘ CH 7.34, d (8.1 Hz) 128.7 C-3 C-2, H-3‘ 5“‘ CH 6.80, d (8.2 Hz) 115.4 C-4“‘ C-1“‘ H-6“‘
2‘/6‘
3‘ CH 6.80, d (8.2 Hz) 116.0 C-2‘, 4‘ C-1‘ C-6‘ H-2‘ 6“‘ CH 7.57, d (8.6 Hz) 130.6 C-1“‘, C-4“‘, C-3“‘, H-5“‘
C-5“‘ 2“‘, 8“‘
C-7“‘
4‘ C 9.87, s 163.2 7“‘ CH 7.58 c 145.3 C-8“‘ H-8“‘
5‘ CH 6.80, d (8.2 Hz) 116.0 C-4‘,6‘ H-6‘ 8“‘ CH 6.41, d (15.9 Hz) 114.3 C-7“‘ H-7“‘
6‘ CH2 7.34, d, (8.1 Hz) 128.7 H-5‘ 9“‘ C=O 166.1
a b c
Measured at 500 MHz, measured at 125 MHz, overlapped signal.
Separations 2023, 10, 76 11 of 21

3.2. Biological Activities of the Main Fractions and Isolates from E. erinaceus
3.2.1. Acute Toxicity Test
Screening of the toxic effect of increased oral doses of the total MeOH extract revealed
that the extract was non-toxic up to 500 mg/kg of body weight. The observation of the
animals revealed no lethal effects and behavioral signs of toxicity at the tested doses,
indicating that LD50 was greater than 50 mg/kg, with low toxicity in short-term use on the
E. erinaceus crude methanol extract. Thus, the dose (500 mg/ kg, body weight) was selected
for the in vivo experiments and the plant was categorized as safe.

3.2.2. In Vivo Anti-Ulcer Activity of Crude Extracts

Based on the biologically guided procedure, the effect of five extracts of E. erinaceus on
the ethanol-induced gastric ulcer rat model was evaluated to select promising fraction(s)
for further phytochemical investigations (Table 4). Oral administration of absolute ethanol
produced multiple mucosal lesions in the rat stomach. The results indicated that pre-
treatment with antodine (the reference standard) and crude E. erinaceus extracts decreased
ethanol-induced gastric mucosal injuries. The percentage of the ulcer protection was
increased significantly (p < 0.05) in rats pre-treated with antodine (20 mg/Kg), MeOH,
n-hexane (Hex), CHCl3 , EtOAc, and the remaining aqueous (ReAq) extracts at a dose
of 500 mg/Kg compared to the ethanol control group (63%, 71%, 73%, 72%, 67%, and
64%, respectively) (Table 4 and Figure 2). Remarkably, the ulcer protection of rats pre-
treated with all tested extracts showed better results than antodine. TNF-α (tumor necrosis
factor-α) and IL-6 (interleukin-6), the proinflammatory cytokines that contribute to various
immunologic and inflammatory responses, were assessed [38,39]. The administration of
ethanol produced an elevation in the stomach contents of TNF-α and IL-6 by 1.3 and 27-folds,
respectively, compared to normal control values (Figures S34 and S35). Treatment with
antodine reduced the stomach contents of TNF-α and IL-6 by 22 % and 55%, respectively, as
compared to the ethanol group. However, the treatment with the different tested extracts
(MeOH, n-Hex, CHCl3 , EtOAc, and ReAq) decreased the stomach contents of TNF-α by
Separations 2023, 10, 76 24%, 25%, 36%, 28%, and 21%, and reduced IL-6 gastric content by 54%, 74%, 9413%, of 81%,
22

and 35%, respectively, as compared to the ethanol group (Table 4).

Figure 2.
Figure 2. Gross
Grossappearance
appearance of of
gastric mucosa
gastric of rats
mucosa treated
of rats with;with;
treated salinesaline
(a), ethanol 1 mL (b),
(a), ethanol etha‐(b),
1 mL
nol + antodine
ethanol 20 mg/kg
+ antodine (c), ethanol+
20 mg/kg MeOHMeOH
(c), ethanol+ extract extract
(d), ethanol+ n‐Hex extract
(d), ethanol+ n-Hex(e), ethanol+
extract CHCl3
(e), ethanol+
extract (f), ethanol+ EtOAc extract (g), and ethanol+ ReAq extract (h), using an oral dose of 500 mg
CHCl3 extract (f), ethanol+ EtOAc extract (g), and ethanol+ ReAq extract (h), using an oral dose of
extract/kg.
500 mg extract/kg.
3.2.3. Histopathological Examination
The control group showed normal structure of the mucosal layer (mu) with glandular
structure and lamina propria, as well as the underlying submucosa (subm) and the muscu‐
laris (mus) (Figure 3a). However, the group with ethanol‐induced ulcers showed focal ne‐
crosis (n), which was detected in the glandular structure of the mucosal layer (Figure 3b)
Separations 2023, 10, 76 12 of 21

Table 4. Effect of the different extracts of E. erinaceus (500 mg/ kg) on ethanol-induced ulcers and on
the gastric contents of TNF-α and IL-6.

Control Ethanol Antodine

Control MeOH Ext. n-Hex Ext. CHCl3 Ext. EtOAc Ext. ReAq Ext.
(Saline) (1 mL/kg) (20 mg/kg)

Ulcer index
(mean ± SED)
0 ± 00 6.43 ± 0.34 2.37 ± 0.14 ab 1.83 ± 0.05 abc 1.77 ± 0.11 abc 1.80 ± 0.04 abc 2.07 ± 0.09 abc 2.30 ± 0.12 ab

% Ulcer 100% – 63% 71% 73% 72% 67% 64%

protection
TNF-α
(pg/mL)
1970 ± 25.69 4614 ± 176.79 a 3610 ± 45.50 ab 3492 ± 71.65 ab 3448 ± 99.42 ab 2948 ± 64.53 abc 3322 ± 194.97 ab 3644 ± 137.54 ab

IL-6 (pg/mL) 5.6 ± 0.51 156.8 ± 3.50 a 70.6 ± 1.29 ab 71.4 ± 7.43 ab 40.2 ± 1.71abc 9.46 ± 1.29 bc 29.8 ± 1.62 abc 101.4 ± 7.43 abc
All the values are presented as means ± standard deviation of the means (SD) and n = 8. Statistical analysis was
carried out by one-way ANOVA followed by HSD Tukey’s multiple comparisons test. a Significantly different
from normal control at p < 0.05. b Significantly different from ethanol at p < 0.05. c Significantly different from
antodine at p < 0.05.

3.2.3. Histopathological Examination

The control group showed normal structure of the mucosal layer (mu) with glandular
structure and lamina propria, as well as the underlying submucosa (subm) and the mus-
cularis (mus) (Figure 3a). However, the group with ethanol-induced ulcers showed focal
necrosis (n), which was detected in the glandular structure of the mucosal layer (Figure 3b)
and associated with inflammatory cells’ (arrow) infiltration of the lamina propria of the
mucosa, as well as edema (o) in the underlying submucosa and later, dilated blood vessels
(Figure 3c). The group with ethanol-induced ulcers treated with reference drug showed few
inflammatory cells infiltrating (arrow) the lamina propria and associated with edema (o) in
the submucosa (Figure 3d). The group of ethanol-induced ulcer rats treated with MeOH ex-
tract showed edema (o), inflammatory cell infiltration (arrow), and dilatated blood vessels
(Figure 3e). The group of ethanol-induced ulcer rats treated with n-Hex extract showed
edema (o) with a few inflammatory cells’ infiltration (arrow) detected in the submucosa
(Figure 3f). The group of rats with ethanol-induced ulcers treated with CHCl3 extract
showed the best result, with no histopathological alteration in the mucosa (mu), submucosa
(subm), muscularis (mus), and serosa (s) as recorded in (Figure 3g). The group of rats with
ethanol-induced ulcers treated with EtOAc extract showed few inflammatory cells’ (arrow)
infiltration, and edema (o) was detected in the submucosa (Figure 3h). The group of rats
with ethanol-induced ulcers treated with ReAq extract showed edema (o), inflammatory
cells’ infiltration (arrow) and dilated blood vessels in the submucosa (Figure 3i).

3.2.4. In Vitro Anti-Inflammatory Activity of Isolated Compounds

In our previous research [12], the CHCl3 extract showed remarkable anti-inflammatory
activity. In the current study, the isolated compounds and their fractions were tested for
enzyme-inhibitory activity on COX-2 and 15-LOX enzymes. A bio-guided fractionation of
the CHCl3 extract revealed that Fraction 6 showed strong activity, with IC50 of 4.51± 0.78
and 12.95 ± 1.23 µg/mL, respectively. In addition, compound E7 isolated from the same
fraction was the most active inhibitor against COX-2 and 15-LOX enzymes, with IC50
of 2.62 ± 0.71 and 5.51 ± 0.76 µg/mL, respectively, compared to the reference standard,
nordihydroguairetic acid (NDGA). Although, compound E3 displayed significant inhibitory
activity against COX-2, with an IC50 of 3.41 ± 0.65 µg/mL. However, compounds E2
and E6 showed moderate inhibitory activity against COX-2 with IC50 of 6.54 ± 0.86 and
8.19 ± 0.93 µg/mL, respectively, (Table 5).
 Separations
Separations 2023, 2023,
10, 7610, 76 14 of 22
13 of 21

Figure
Figure 3. 3.Effect
Effect of
of the
the various
variousextracts of E.
extracts E. erinaceus
of erinaceus on the
onhistological structure
the histological of the gastric
structure of themu‐
gastric
cosa ofofdifferent
mucosa differenttreated rats’
treated groups
rats’ stained
groups with with
stained H & EHstain
& E(x40);
stain(a)
(×control
40); (a)group
controlof group
normalofrats;
normal
(b,c) ethanol‐induced ulcer group; (d) ethanol‐induced group + antodine; (e) ethanol‐induced group
rats; (b,c) ethanol-induced ulcer group; (d) ethanol-induced group + antodine; (e) ethanol-induced
+ MeOH ext; (f) ethanol‐induced group + n‐Hex. ext. (g) ethanol‐induced group + CHCl3 ext. (h)
group + MeOH ext;
ethanol‐induced (f) ethanol-induced
group group + n-Hex.
+ EtOAc ext. (i) ethanol‐induced ext. +(g)
group ethanol-induced
ReAq ext. mucosa = mu,group + CHCl3
submu‐
ext.cosa
(h)=ethanol-induced
subm, muscularis group + EtOAc
= mus, serosa ext. (i)
= s, focal ethanol-induced
necrosis = n, oedema =group + ReAq ext.
o, inflammatory cellsmucosa
= arrow.= mu,
submucosa = subm, muscularis = mus, serosa = s, focal necrosis = n, oedema = o, inflammatory
cells = arrow.
Separations 2023, 10, 76 14 of 21

Table 5. IC50 and binding free energies of cyclooxygenase-2 (COX-2) and 15-lipoxygenase (15-LOX)-
inhibitory activities of the selected sub-fractions and isolated compounds (E1–E7) from E. erinaceus.

COX-2 a 15-LOX a
Sample ID Binding Free Energy Binding Free Energy
IC50 (µg/mL) IC50 (µg/mL)
(kcal/mol) (kcal/mol)
Fr. 3 35.39± 1.94 — 46.27 ± 3.74 —
Fr. 4 12.39 ± 1.27 — 21.17± 1.41 —
Fr. 5 3.26 ± 0.69 — 3.02 ± 0.49 —
Fr. 6 4.51± 0.78 — 12.95 ± 1.23 —
E1 49.61± 2.75 −8.3 49.89 ± 3.12 −6.5
E2 8.19 ± 0.93 −9.0 13.06 ±0.98 −6.7
E3 3.41± 0.65 −6.1 10.05± 0.96 −6.0
E4 15.52 ± 1.41 −7.4 68.73 ± 5.76 −7.0
E5 37.0± 2.59 −7.1 47.45 ± 3.88 −6.6
E6 6.54 ± 0.86 −7.6 16.11 ± 1.27 −6.3
E7 2.62 ± 0.71 −7.7 5.51 ±0.76 −9.2
NDGA b 1.42± 0.23 — 1.71± 0.27 −–
Celecoxib — — — −6.3
RS7 — −6.2 — —
a b
Samples were analysed in triplicate (n = 3) and expressed as mean ± standard deviation. NDGA: nordihy-
droguairetic acid (reference standard drug). RS7 = (2E)-3-(2-oct-1-yn-1-ylphenyl)acrylic acid (reference ligand
ID:1LOX); Celecoxib (reference ligand ID:3LN1); [22,23]. For detailed interacting amino acid residues, see the
Supplementary File.

3.3. Docking Study

3.3.1. PASS and ADME Predictions of the Isolated Compounds
The SMILES format of isolated compounds was chosen using the Marvinsketch pro-
gram and simulated by using the PASS and ADME prediction web tools [40–42]. The
PASS tool predicted several biological activities, such as anti-inflammatory, anticancer,
antioxidant, anti-ulcerative, and antiosteoporosis/antiarthritic activities. The isolated com-
pounds (E1–E7) displayed significant “Pa” values ranging from (0.595–0.749; 0.524–0.960;
0.240–0.991; 0.406–0.749; and 0.296–0489) potential, respectively, for the above-mentioned
biological activities (Table 6). Therefore, all compounds exhibited significant excellent
anti-inflammatory, antineoplastic, and good antioxidant properties alongside excellent anti-
ulcerative and antiosteoporosis/antiarthritic activities, except for compounds E5 and/or
E6, from data analysis (Table 6). The ADME prediction web tool was used to predict
the physicochemical, pharmacokinetic, and drug-likeness properties of the isolated com-
pounds. The results showed that tested compounds (E1–E7) showed a bioactivity score
range of 0.17–0.56, which fulfilled all the drug-likeness rules without any violations; the
synthetic accessibility range was 2.96–7.93, which showed an explicit synthetic route. The
lipophilicity values of the compounds showed that they have variable solubility in water.
In addition, skin permeation, absorption, distribution, and metabolism were analyzed by
using Swiss-ADME software, recorded in Table 6. The BOILED-Egg method was used
to predict the ability of the tested compounds for GI absorption and passive diffusion
through the blood–brain barrier (BBB) [43]. The BOILED-Egg prediction results (Figure 4)
showed that compounds E3–E6 have GI absorption properties with poor diffuse through
the BBB, and that they have good permeability value for skin permeability parameters (log
Kp) [44]. Compounds E1–E4 & E7 showed no response to glycoprotein (P-gp) which has
a significant role in drug absorption and distribution. Compound E4 inhibited CYP1A2,
CYP2D6, and CYP3A4, and E5 inhibited CYP3A4, resulting in drug–drug interactions
Separations 2023, 10, 76 15 of 21

and adverse effects [40]. The findings revealed that four out of the seven compounds
fulfilled the oral drug ability of Lipinski’s rule of five (RO5), while three slightly met the
criteria of RO5. Conversely, two of the compounds were predicted to be mutagen, while
the remaining two are predicted to be safe for the body.

Table 6. In silico physicochemical and pharmacokinetics of the major compounds E1–E7.

Predictive E1 E2 E3 E4 E5 E6 E7
Parameters
Pass prediction (Pa/Pi) a

Anti-
inflammatory 0.736/0.012 0.749/0.010 0.595/0.033 0.644/0.024 0.685/ 0.018 0.599/0.032 0.736/0.012

Antiosteoporosis
0.421/0.020 * 0.489/0.015 * 0.456/0.059 ** 0.468/ 0.017 * - 0.481/0.016 * 0.296/0.040 *
*/Antiarthritic **
Anti-ulcerative 0.610/0.010 0.574/0.013 0.406/0.043 0.495/0.024 - - 0.749/0.004
Antineoplastic 0.960/0.004 0.960/0.004 0.661/0.033 0.774/0.015 0.524/0.064 0.695/0.027 0.850/0.007
Oxygen scav-
0.240/0.039 0.283/0.027 0.376/0.110 0.732/0.004 0.452/0.009 0.379/0.014 0.991/0.001
enger/antioxidant
ADME Prediction b

Physicochemical parameters
TPSA (0 A2 ): 26.30 Å2 20.23 Å2 94.83 Å2 90.90 Å2 122.52 Å2 99.38 Å2 192.44 Å2
Molar refractivity 144.88 135.14 70.94 73.99 127.20 165.14 145.06
Drug-likeness prediction
Bioavailability 0.55 0.55 0.56 0.55 0.55 0.55 0.17
score
Synthetic
5.61 5.40 4.00 2.96 6.37 7.93 5.75
accessibility
Absorption prediction
Log S (ESOL) −8.73 −8.24 −1.54 −3.94 −3.52 −7.26 −4.75
Consensus Log 7.51 7.11 1.06 2.11 2.37 5.30 1.63
Po/w
Moderately
Solubility class Poorly soluble Poorly soluble Very soluble Soluble Soluble Poorly soluble
soluble
Distribution prediction/pharmacokinetics
Log Kp (skin
permeation, −2.27 −2.42 −7.63 −5.80 −7.87 −4.86 −8.13
cm/s)
GI absorption Low Low High High High High Low
BBB permeant No No No No No No No
Metabolism prediction
P-gp substrate No No No No Yes Yes No
CYP1A2,
CYP2C19, Yes, No, No, All No, except
CYP2C9, CYP2D6, No No No Yes, Yes No No
and CYP3A4 Yes (CYP3A4)
inhibitors
a PASS prediction web tool [41], b SwissADME web tool [42], where “Pa” is probable activity, “Pi” is probable
inactivity; * probable antiosteoporosis activity; ** probable antiarthritic activity; “Å2 ” polar surface area and
“TPSA” Topological polar surface area; Taraxasterol acetate (E1), Taraxasterol (E2), Erinaceolic acid (E3), Apigenin
(E4), Erinaceoside (E5), Stigmasterol-3-O-β-D-glucoside (E6), and Speranskoside (E7).

3.3.2. In Silico Molecular Docking Study of Isolated Compounds

To understand the inhibitory effects of the secondary metabolites from E. erinaceus on
the pro-inflammatory enzymes, compounds (E1–E7) isolated from the plant were docked
on nonhuman counterparts of COX-2 (PDB ID: 3LN1), and 15-LOX (PDB ID: 1LOX).

Interactions with COX-2

All tested compounds interacted with the COX-2 enzyme and docked inside the 3LN1
receptor. Most of these compounds exhibited binding poses that were similar to that of
celecoxib, through binding with the formation of an H-bond with Gln178, His75, and Tyr341
Separations 2023, 10, 76 16 of 21

residues, except E2 and E5 [45,46]. However, they displayed hydrophobic interactions with
Separations 2023, 10, 76 Arg106, Arg499, and Ser339 residues, except for E1 and E2. The protein–ligand interactions
17 of 22
observed in the representative docking poses of tested compounds are summarized in
Table S6 and in Figure 5, Figures S36 and S37.

4. Bioavailability
Figure 4.
Figure Bioavailability radar
radar representations
representationsand
andpredicted
predictedBOILED-Egg
BOILED‐Eggdiagram
diagramofofcompounds
compounds
E1–E7.

3.3.2. In Silico Molecular Docking Study of Isolated Compounds

To understand the inhibitory effects of the secondary metabolites from E. erinaceus
on the pro‐inflammatory enzymes, compounds (E1‐E7) isolated from the plant were
docked on nonhuman counterparts of COX‐2 (PDB ID: 3LN1), and 15‐LOX (PDB ID:
1LOX).
Interactions with COX‐2
All tested compounds interacted with the COX‐2 enzyme and docked inside the

Separations 2023, 10, 76
idues (Table S6, Figure 5a,b) [45–47].
On the other hand, compounds E1, E3–E6 also yielded good binding free energies
(kcal/mol) within COX‐2, showing H‐bond interactions with the His75, His337, 342,
Gln178 Asn567, Gln336, Gly340, Pro177, and Thr79 residues. While E2 did not form hy‐
drogen bonds with the crucial amino acids in the catalytic pocket, E2–E5 compounds did
not form π ‐bonds with crucial amino acids at the hydrophobic binding site (Table S6,17 of 21
Figure S36).

Figure
Figure 5. Three‐dimensional(3D)
5. Three-dimensional (3D)and
and two-dimensional
two‐dimensional (2D) molecular
(2D) binding
molecular interactions
binding of theof the
interactions
dual inhibitor compound E7 with: (a,b) cyclooxygenase‐2, COX‐2 (PDB: ID 3LN1), (Dimensions
dual inhibitor compound E7 with: (a,b) cyclooxygenase-2, COX-2 (PDB: ID 3LN1), (Dimensions
X:25.000, Y:21.2305, Z:24.7079); and (c,d) 15‐lipooxygense, 15‐LOX (PDB ID: 1LOX), (dimensions
X:25.000, Y:21.2305,
X:20.6138, Z:24.7079);
Y: 18.9027, and (c,d) 15-lipooxygense, 15-LOX (PDB ID: 1LOX), (dimensions
Z:26.9761).
X:20.6138, Y: 18.9027, Z:26.9761).

Compound E7 had the highest consensus score value (−9.2 Kcal/mol) with the forma-
tion of six H-bonds between the hydroxyl groups with His75 and Tyr341, Asp501, His337,
Pro177, and Thr79 amino acids. Benzene rings of the coumaroyl and flavanone moieties
adopt poses with the formation of amide-π stacked and π-σ bonds with Gln336 and Pro500,
respectively. The hydrophobic affinity of the residues was found in the hydrophobic chan-
nel of the active site, which interacted with Gln178, His75, and Ser339 residues (Table S6,
Figure 5a,b) [45–47].
On the other hand, compounds E1, E3–E6 also yielded good binding free energies
(kcal/mol) within COX-2, showing H-bond interactions with the His75, His337, 342, Gln178
Asn567, Gln336, Gly340, Pro177, and Thr79 residues. While E2 did not form hydrogen
bonds with the crucial amino acids in the catalytic pocket, E2–E5 compounds did not form
π -bonds with crucial amino acids at the hydrophobic binding site (Table S6, Figure S36).

Interactions with 15-LOX

The results of docking with 15-LOX revealed that the analyzed compounds (E1–
E7) showed binding affinity (−8.3, −9.0, −6.1, −7.4, −7.1, −7.6, and −7.7 Kcal/mol,
respectively) greater than or the same as the co-crystalized ligand, RS7 (2E)-3-(2-oct-1-yn-1-
ylphenyl)acrylic acid, −6.2 Kcal/mol), as shown in Table 5.
Compound E7 was placed in the active center of the enzyme in a curved conformation
with the coumaroyl moiety oriented in the vicinity of Arg403, Gly407, Leu408, and Leu597,
and the ring B of naringenin moiety placed near Phe175 (Table S6, Figure 5c,d). One H-bond
Separations 2023, 10, 76 18 of 21

was formed between the H atom of the sugar group linked to the flavanone moiety and
the O atoms of the peptide bonds of Arg403, and the second H-bond was formed between
the carbonyl group of the coumaroyl moiety with H atoms of Gly407; meanwhile, two
hydrogen bonds and two benzene ringsparticipate in π-π interactions with Arg403, Gly407,
Leu408, Leu597, and Phe175. In addition, hydrophobic interactions with Ala404, and Ile414
also contribute to complex stabilization, and confirmed the greatest inhibitory activity of
the in vitro study of 15-LOX, with an IC50 of 5.51 ±0.76 (Tables 5 and S6, Figure 5c,d).
The hydrophilic compound E5 showed a great binding affinity with Gly407, Gly598,
and Gln596, as inferred by the high docking score of −7.1 Kcal/mol when compared to the
control, RS7 (−6.2 Kcal/mol). This could be explained by the presence of three H-bonds
of two carbonyl groups (C=O) and one hydroxy (-OH) group. Additionally, the presence
of the glucose and isovalerate moieties improved the capacity for interaction with the
hydrophobic binding site of 15-LOX with Ala404, Arg403, Gly407, Ile663, and Leu408 as
shown in Table S6 and Figure S37.
Additionally, the active compound E3 had the same binding energy (−6.1 Kcal/mol)
as the co-crystalized ligand (RS7= −6.2 Kcal/mol) in the 15-LOX active site. This can be
explained by the two hydrogen bonds formed between the carboxylic group with Arg403
and Asn406, and the presence of the hydrophobic interactions with Ala404, His545, Ile414,
Ile663, Leu408, Leu597, and Phe175. In addition, no π-π interactions were observed in
this case, which explained the low binding free energy of E3, and consequently the in vitro
15-LOX enzyme assay results (IC50 10.05± 0.96 µg/mL) (Table 5 and Table S6, Figure S37).
Regarding the virtual interactions, E1, E2, and E6 also showed interaction with the hy-
drophobic binding site of the 15-LOX enzyme through the presence of acetoxy (-OCOCH3 ),
hydroxy (-OH), and glucose-methyl groups (-CH3 ), respectively (Table S6, Figure S37).
These results were comparable to the previously published data of the arachidonic binding
site which revealed the importance of the binding interactions of the acidic ends of the
tested compounds with Arg403, Gly407, and Leu408, and the absence of catalytic residues
353–361 for the 15-LOX-inhibitory activity [23,48].

4. Conclusions
In this study, two undescribed terpenoid derivatives (erinaceolic acid, E3 and eri-
naceoside, E5), a rare flavanone glycoside derivative (speranskoside, E7), together with
two known taraxastane-type triterpenes (taraxasterol acetate, E1 and taraxasterol, E2),
one flavone (apigenin, E4), and one steroidal glycoside (stigmasterol-3-O-β-D-glucoside,
E6), were isolated and characterized by spectroscopic methods, including 2D NMR and
HRESIMS experiments. The total extract and successive fractions of E. erinaceus, espe-
cially the CHCl3 fraction, alleviated ethanol-induced gastric ulcers in rats. The different
extracts of E. erinaceus are shown to be highly safe for human use as anti-inflammatory
and antiulcerogenic remedies. The results of the in vitro and in silico studies indicated
that the new molecules (E3 and E5) and the other isolated known compounds (E1, E2,
E4, E6, and E7) could be promising inhibitors of COX-2 and 15-LOX enzymes and may
contribute to the obtained in vivo antiulcer effect. However, more pharmacological and
pharmacokinetic experiments are needed to establish their use in the prophylaxis and
treatment of gastric ulcers.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/separations10020076/s1, Tables S1 and S4: NMR spectroscopic
data for the compounds E1 and E2 and E6.; Tables S2, S3, and S5: HR-ESI-MS spectral data of
compounds E3, E5, and E7.; Table S6: Docking of compounds against 15-LOX and COX-2 enzymes.;
Flowchart S1-S3: Extraction, fractionation, and purification procedures of compounds (E1–E7).;
Figure S1–S33: 1D- and 2D NMR, HR-ESI-MS spectral data of compounds E1–E7.; Figure S34–S34:
Effect of the tested extracts on gastric contents of TNF-α and IL-6.; Figure S36: 3D and 2D molecular
interactions of E1-E7 with COX-2.; Figure S37: 3D and 2D molecular interactions of E1, E2, E4, and E6
compounds with 15-LOX enzyme. The full spectral data, including the NMR and HRESIMS spectra,
and the results of the in silico study. References [49,50] are cited in the Supplementary Material.
Separations 2023, 10, 76 19 of 21

Author Contributions: Conceptualization, E.A.-S. and M.M.E.-S.; fractionation and isolation of the
compounds, S.H.S.; Identification of the compounds, S.H.S., F.M.A.B. and E.A.-S.; Molecular docking
and writing of original draft, S.H.S.; Writing, review and editing, E.A.-S., M.M.E.-S., O.D.E.-G., and
F.M.A.B.; in vitro & in vivo biological activities performance, A.I.F., M.H.A. and S.H.S. All authors
have read and agreed to the published version of the manuscript.
Funding: This study is supported via funding from Prince Sattam bin Abdulaziz University project
number (PSAU/2023/R/1444).
Data Availability Statement: The data presented in this study are available in the Supplementary
Materials at https://www.mdpi.com/article/10.3390/separations10020076/s1.
Acknowledgments: The authors acknowledged Amani Awaad, for her kind cooperation in suggest-
ing the title plant for this study, as well as Abeer A. Salama for performing the biological study at
Pharmacology Department, National Research Center, Dokki, Egypt.
Conflicts of Interest: The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this paper.

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