separations

Article
Phytochemical, Antimicrobial, Antioxidant, and In Vitro
Cytotoxicity Evaluation of Echinops erinaceus Kit Tan
Sherouk Hussein Sweilam 1,2 , Fatma M. Abdel Bar 1,3 , Ahmed I. Foudah 1 , Mohammed H. Alqarni 1 ,
Nouran A. Elattal 4 , Omayma D. El-Gindi 2 , Moshera M. El-Sherei 5 and Essam Abdel-Sattar 5, *

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, Cairo 11829, Egypt
3 Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
4 Department of Chemistry of Natural and Microbial Products, National Research Center, Dokki,
Giza 12622, Egypt
5 Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562, Egypt
* Correspondence: [email protected]

Abstract: Wild plants are used by many cultures for the treatment of diverse ailments. However,
they are formed from mixtures of many wanted and unwanted phytochemicals. Thus, there is a
necessity to separate the bioactive compounds responsible for their biological activity. In this study,
the chemical composition as well as antimicrobial and cytotoxic activities of Echinops erinaceus Kit Tan
(Asteraceae) were investigated. This led to the isolation and identification of seven compounds, two
of which are new (erinaceosin C3 and erinaceol C5), in addition to methyl oleate (C1) and ethyl oleate
(C2), loliolide (C4), (E)-p-coumaric acid (C6), and 5,7,30 ,50 -tetrahydroxy flavanone (C7). The structures
Citation: Sweilam, S.H.; Abdel Bar,
of the isolated compounds were elucidated by 1D, 2D NMR, and HR-ESI-MS. The methanol extract
F.M.; Foudah, A.I.; Alqarni, M.H.;
showed the highest antimicrobial activity among the tested extracts and fractions. The n-hexane and
Elattal, N.A.; El-Gindi, O.D.;
El-Sherei, M.M.; Abdel-Sattar, E.
EtOAc extracts showed remarkable antimicrobial activity against B. subtilus, P. aeruginosa, E. coli, and
Phytochemical, Antimicrobial, C. albicans. A cytotoxicity-guided fractionation of the most bioactive chloroform extract resulted in the
Antioxidant, and In Vitro isolation of bioactive compounds C1/C2, which showed significant cytotoxicity against HCT-116 and
Cytotoxicity Evaluation of Echinops CACO2 cell lines (IC50 24.95 and 19.74 µg/mL, respectively), followed by compounds C3 (IC50 82.82
erinaceus Kit Tan. Separations 2022, 9, and 76.70 µg/mL) and C5 (IC50 99.09 and 87.27 µg/mL), respectively. The antioxidant activity of
447. https://doi.org/10.3390/ the bioactive chloroform fractions was screened. Molecular docking was used to explain the results
separations9120447 of the antimicrobial and anticancer activities against five protein targets, including DNA gyrase
Academic Editor: Marcello Locatelli topoisomerase II, enoyl-acyl carrier protein reductase of S. aureus (FabI), dihydrofolate reductase
(DHFR), β-catenin, and human P-glycoprotein (P-gp).
Received: 1 December 2022
Accepted: 12 December 2022
Keywords: Echinops erinaceus; antimicrobial; cytotoxicity; pseudoguaiane sesquiterpene; abscisic
Published: 16 December 2022
alcohol derivative; phenolic compounds
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. 1. Introduction
Plants of the family Asteraceae demonstrate significant therapeutic applications be-
cause of their unique and diverse pool of secondary metabolites. The reported biological
Copyright: © 2022 by the authors.
activities include antioxidant, antiproliferative, anti-ulcer, and anti-inflammatory activities.
Licensee MDPI, Basel, Switzerland.
They are mainly due to their wide range of phytochemicals, such as phenolics [1], sesquiter-
This article is an open access article pene lactones [2], alkaloids [3,4], and triterpenes [5,6]. Traditionally, the plants of the genus
distributed under the terms and Echinops (Asteraceae) are used to relieve gastrointestinal disturbances [7], kidney inflam-
conditions of the Creative Commons mation [8], microbial infections, and pain [9,10]. In addition, other reported biological
Attribution (CC BY) license (https:// properties include hepatoprotective [11], antifertility [12], analgesic, antipyretic, wound-
creativecommons.org/licenses/by/ healing, anthelmintic [13,14], and insecticidal properties [9,15]. Echinops spp. contains
4.0/). thiophenes, terpenoids (such as sesqui- and triterpenoids), phenolics (such as flavonoids,

Separations 2022, 9, 447. https://doi.org/10.3390/separations9120447 https://www.mdpi.com/journal/separations

Separations 2022, 9, 447 2 of 18

coumarins, phenylpropanoids, and lignans), alkaloids, and essential oils [9,16]. Echinops
erinaceus Kit Tan is an annual herbaceous plant strictly distributed in the Arabian Penin-
sula and is well-known in Saudi Arabia and Yemen under the name of “Alkana’a, Kanab,
and Hawa elghool”. Reviewing the literature data, nothing was reported regarding the
traditional uses of the plant. Our research group previously reported the phytochem-
ical screening, antioxidant, and in vitro anti-inflammatory activities of E. erinaceus [17].
However, research studies that describe the phytochemical composition of this plant are
still scarce.
Cancer is one of the most common diseases that cause death worldwide with an
increased number of new cases every year. The common treatment for cancers is chemother-
apy. Nowadays, patients are suffering from multidrug resistance (MDR), a phenomenon
whereby cells confer drug resistance to structurally and functionally unrelated compounds.
MDR may be a consequence of reduced drug influx, increased drug efflux, activation
of detoxifying systems, activation of DNA repair mechanisms, evasion of drug-induced
apoptosis, etc. [18]. One of the most common mechanisms in MDR is often associated with
decrease in cellular drug accumulation mediated by MRP1 (ABCC1) and/or P-glycoprotein
(P-gp, ABCB1). To overcome MDR, MRP1 and/or P-gp proteins inhibitors are used to
block the transport function might be potentially used [19]. There is an urgent need for
discovering new natural products against life-threatening multidrug-resistant microbial
pathogens [20] and chemotherapy.
The current study aimed at bioguided isolation, structure elucidation, and in vitro
biological evaluation of E. erinaceus. The in vitro biological evaluation targeted the cytotoxic
activity against HCT-116 (colon carcinoma), CACO2 (human colorectal intestinal carci-
noma) cell lines, and its selectivity was assessed using the normal mammalian cell line,
WI-38 (human lung fibroblast) using crystal violet assay. The antimicrobial activities of
the fractions and the isolated compounds were performed on six pathogens, including
two Gram-positive (viz., B. subtilus and MRSA), two Gram-negative bacteria (viz., E. coli,
P. aeruginosa), a fungus (viz., A. niger), and a yeast-like pathogen (viz., C. albicans). More-
over, to reveal the potential molecular mechanisms responsible for the multi-biological
activities of the isolated compounds, molecular docking experiments were conducted
against P-glycoprotein (P-gp), a key protein in MDR of anticancer drugs [21], and human
dihydrofolate reductase (DHFR) involved in malignancies and multidrug-resistance of
microbial pathogens [22], in addition to DNA gyrase topoisomerase II and enoyl-acyl
carrier protein reductase of S. aureus (FabI) as targets for bacteria and β-catenin as a target
for cancer.

2. Materials and Methods

2.1. Plant Material and Extraction
The flowering aerial parts of E. erinaceus Kit Tan were collected from Riyadh region,
Saudi Arabia, in March 2018 [17]. The plant material was authenticated by Mohamed Abdel-
Fattah, a taxonomist and botanist, the botanical garden of the Department of Botany and
Microbiology, College of Science, King Saud University. A verifier specimen (ID: 23.6.19.1-5)
was placed at the local herbarium of the Pharmacognosy Department, Faculty of Pharmacy,
Cairo University. The powdered shade-dried plant material was extracted and fractionated
according to the method reported by Sweilam et al. (2021). The crude MeOH extract, its
fractions, and the isolated compounds (C1–C7) from CHCl3 fraction were subjected to
in vitro cytotoxic and antimicrobial investigations.

2.2. Isolation and Purification of Compounds from the CHCl3 Fraction

The powdered plant material (4 kg) was extracted by cold maceration with MeOH
(5 × 10 L). The obtained extract was evaporated by a rotary evaporator (Büchi, Lugano,
Switzerland) to a semisolid consistency (650 g) which was suspended in water and frac-
tionated successively with solvents viz. n-hexane (Hex), chloroform (CHCl3 ), and ethyl
acetate (EtOAc), to give 150 g, 50 g, and 60 g, respectively [17]. The CHCl3 fraction (38 g)
Separations 2022, 9, 447 3 of 18

was chromatographed on a Si gel column CC (170 × 5 cm2 ) and eluted with a mixture
of CHCl3 /MeOH (100/0 to 70/30). The effluent was monitored using TLC on Si gel
GF245 plates and visualized by spraying with 10% vanillin-H2 SO4 reagent (Flowchart
S1). Fraction-1 (1.0 g) eluted with CHCl3 (100%) was further purified on a Si gel CC
(50 × 1 cm2 ) with gradient elution (EtOAc in n-hexane; 1–10%) to afford subfraction-1-II
(500 mg). The latter was purified on an MPLC RP-18 column (isopropanol–water, 6:4
to 10:0) to obtain compounds C1/C2 as an unresolved mixture (methyl oleate C1/ethyl
oleate C2, 9 mg). Fraction-3 (1.0 g) was subjected to a Si gel CC (65 × 2 cm2 ) and eluted
with EtOAc in n-hexane (10 to 50%) to obtain subfraction-3 I-IX. Subfraction-3-II (120 mg)
and subfraction-3-III (200 mg) were purified by chromatography onto an MPLC RP-18
column (MeOH-H2 O, 1:1 and 3:7, respectively) to give compounds C3 (erinaceosin, 8 mg)
and C4 (loliolide, 4.5 mg), respectively. Fraction-4 was subjected to purification on an
MPLC RP-18 column (MeOH-H2 O, 3:7) followed by a Sephadex LH-20 column to afford
compound C5 (erinaceol, 3 mg). In addition, Fraction-5 (1.5 g) gave subfraction-5-I (65 mg)
and subfraction-5-II (36 mg) upon chromatography on a Si gel column (EtOAc in n-hexane;
10 to 100). Purification of subfraction-5-I and subfraction-5-II on Sephadex LH-20 columns
(MeOH-CH2 Cl2 , 1 to 10) resulted in the isolation of compounds C6 (E-p-coumaric acid,
4 mg) and C7 (5,7,30 ,50 -tetrahydroxy flavanone, 3 mg), respectively (Flowchart S1).

2.3. In Vitro Cytotoxicity Assay

2.3.1. Materials and Cell Lines
Mammalian cell lines HCT-116 cells (human colon cancer cell line), CACO2 cells
(human colorectal intestinal carcinoma), and WI-38 cells (human lung fibroblast normal
cells) were obtained from the American Type Culture Collection (ATCC, Rockville, MD,
USA). Dimethyl sulfoxide (DMSO), crystal violet, and trypan blue dye were purchased
from Sigma (St. Louis, MO, USA). Fetal Bovine serum, RPMI-1640, HEPES buffer solution,
L-glutamine, gentamycin, and 0.25% Trypsin-EDTA were purchased from Lonza (Verviers,
Belgium). Crystal violet stain (1%) made from 0.5% (w/v) crystal violet and 50% MeOH was
then brought to volume with dd.H2 O and filtered through a Whatmann No.1 filter paper.

2.3.2. Cell Culture Condition and Propagation

The HCT-116 and CACO2 cells were propagated in RPMI-1640 medium, and WI-38 cells
were propagated in Dulbecco’s modified Eagle’s medium (DMEM), which was supple-
mented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer, and
50 µg/mL gentamycin. All cells were maintained at 37 ◦ C in a humidified atmosphere with
5% CO2 and were subcultured two times a week [23–25].

2.3.3. Cytotoxicity Evaluation Using Viability Assay

The cells were seeded in a 96-well plate at a cell concentration of 1 × 104 cells per well
in 100 µL of growth medium. Fresh medium containing different concentrations of the
test sample was added after 24 h of seeding. Two-fold serial dilutions of the tested sample
were added to confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter
plates (Falcon, NJ, USA) using a multichannel pipette. The microtiter plates were incubated
at 37 ◦ C in a humidified incubator with 5% CO2 for a period of 48 h. Three wells were
used for each concentration of the test sample. Control cells were incubated without a test
sample and with or without DMSO. The low percentage of DMSO present in the wells
(max 0.1%) was found not to affect the experiment. After incubation of the cells at 37 ◦ C,
various concentrations of samples were added, and the incubation was continued for 24 h,
and viable cells’ yield was determined by a colorimetric method. All experiments were
carried out in triplicate [23–25].
The 50% inhibitory concentration (IC50 , the concentration required to cause toxic
effects in 50% of cancer cells) and the cytotoxic concentration (CC50 , the concentration
required to cause toxic effects in 50% of normal cells) were determined from graphic plots
Separations 2022, 9, 447 4 of 18

of the dose–response curve for each concentration using Graphpad Prism software (San
Diego, CA, USA).

2.4. In Vitro Antimicrobial Activity

The antimicrobial activity of E. erinaceus extracts was tested by the agar well diffusion
method [26] against six micro-organisms (Bacillus subtilus, MRSA, Pseudomonas aeruginosa,
and Escherichia coli on nutrient agar, and Candida albicans, and Asperigllus niger on potato
dextrose agar, PDA). In this experiment, all extracts were dissolved in MeOH (200 µg/mL),
and 50 µL of the prepared sample solution was added to each well, separately in each case.
The diameter of inhibition zone (DIZ) was measured.

2.5. In Vitro Antioxidant Effect

The in vitro antioxidant activity of fractions 3, 4, and fraction 5 of the CHCl3 extract
(Flowchart S1) was established in accordance with Burits and Bucar [27]. In brief, 50 µL of
the sample solution (200 µg/mL) was added to 100 µL of 2,2-diphenyl-1-picrylhydrazyl
(DPPH) methanolic solution (0.1%). After a period of 50 min incubation in the dark,
absorbance was measured at 517 nm against the blank. The inhibition percentage of DPPH
free radicals (I) was as follows:

I (%) = (A blank − B sample/A blank) × 100

where A (blank) is the absorbance of control (containing all reagents, except the test
compound), and B (sample) is the absorbance of the test sample; ascorbic acid is used as a
standard drug.

2.6. In Silico Studies of the Isolated Compounds

2.6.1. PASS and ADME Predictions
The isolated compounds (Figure 1), namely, methyl oleate (C1), ethyl oleate (C2), eri-
naceosin (C3), loliolide (C4), erinaceol (C5), (E)-p-coumaric acid (C6), and 5,7,30 ,50 -tetrahydroxy
flavanone (C7), were drawn by MarvinSketch program and simulated by the Prediction
of Activity Spectra for Substances (PASS) and Absorption, Distribution, Metabolism, and
Elimination (ADME) prediction web tools. The isolated compounds were analyzed by
SwissADME online free site for prediction of physicochemical properties, drug likeness,
solubility, and pharmacokinetics [28–30].

2.6.2. Molecular Docking Analysis

Docking analyses of the isolated compounds were accomplished to understand the
antibacterial and anticancer activities. For antibacterial activity, DNA gyrase topoisomerase
II (E. coli) enzyme (PDB ID: 1KZN) [31] and enoyl-acyl carrier protein reductase of S. aureus,
FabI (PDB ID: 3GNS) [32] were used as the target proteins. For anticancer activity, β-catenin
in complex with compound 6 (PDB ID: 7AFW) [33] was used. For both activities, dihydro-
folate reductase (DHFR) (PDB ID: 4M6J) [34] has been chosen as the target protein. The
study of the MDR of the anticancer and antimicrobial candidates, the human P-gp (PDB
ID: 6C0V) [21], were downloaded from PDB (https://www.rcsb.org (accessed on 1 Decem-
ber 2022)). The downloaded proteins were prepared by removing water and any unwanted
residual matter and adding non-hydrogen atoms by PyMOL 2.3. The PyRx Autodock
Vina (Scripps Research, La Jolla, CA, USA) was utilized for these in silico studies. The
co-crystalized ligands, including clorobiocin, triclosan, 3-[(24-methyl-5-oxidanylidene-2,3-
dihydro-1,4-benzoxazepin-2-yl]benzenecarbonitrile (R9Q), ciprofloxacin, and methotrexate
for studying the comparative binding affinity to the target proteins, were downloaded and
saved in 3D SDF format.
 Separations 2022, 9, x FOR PEER REVIEW 5 of 18

Separations 2022, 9, 447 5 of 18

comparative binding affinity to the target proteins, were downloaded and saved in 3D
SDF format.

Figure 1.
Figure 1. Structures
Structures of
ofisolated
isolatedcompounds
compoundsfrom Echinops
from erinaceus
Echinops (C1,(C1,
erinaceus C2, C2,
C3–C7).
C3–C7).

3. Results and Discussion

3. Results and Discussion
3.1. Identification of the Isolated Compounds
3.1. Identification of the Isolated Compounds
The spectral data of the known compounds (C1, C2, C4, C6, and C7) are recorded in
The spectral data of the known compounds (C1, C2, C4, C6, and C7) are recorded
Tables S1–S4, while the spectra of all isolated compounds (C1–C7) are displayed in Fig-
in Tables S1–S4, while the spectra of all isolated compounds (C1–C7) are displayed in
ures S1–S37.
Figures S1–S37.
3.1.1. Identification of Compounds C1 and C2
3.1.1. Identification of Compounds C1 and C2
The mixture of compounds C1 and C2 was identified as a mixture of two esterified
The mixture of compounds C1 and C2 was identified as a mixture of two esterified
mono-unsaturated long-chain fatty acids using 1D, 2D NMR, and ESI-MS data (Figure 1,
mono-unsaturated long-chain fatty acids using 1D, 2D NMR, and ESI-MS data (Figure 1,
Table S1) [35,36]. It is worth noting that methyl oleate (C1) has been isolated before from
Table S1) [35,36].
the Echinops genusIt[37];
is worth noting
however, thisthat
is themethyl oleate
first report on(C1) has been isolated
the identification of ethylbefore
oleatefrom
the Echinops genus [37]; however, this is the first report on the identification
(C2) from the Echinops genus, although it was reported before in the Asteraceae family of ethyl oleate
[38].from the Echinops genus, although it was reported before in the Asteraceae family [38].
(C2)

3.1.2.
3.1.2. Identification ofCompound
Identification of CompoundC3
C3
According
According to to HR-ESI-MS,
HR-ESI-MS,compound
compoundC3 C3hadhada amolecular
molecular formula
formula of of
C15CH15 H based
24O224
O2 based
on the ion peak at m/z 237.1859 [M + H, 1.5%] (calcd. 237.1855) and 219.1753 [M + − OH,
on the ion peak at m/z 237.1859 [M+H, 1.5%] (calcd. 237.1855) and 219.1753 [M+−OH, 85%]
85%]
(calcd.(calcd. 219.1749).
219.1749). The NMR Thespectral
NMR spectral data of compound
data of compound C3 (TableC3 1) (Table 1) were consistent
were consistent with
with the basic
the basic skeleton
skeleton of pseudoguaiane
of pseudoguaiane sesquiterpenes,
sesquiterpenes, except forexcept for the presence
the presence of the
of the α, β-
unsaturated
α, β-unsaturatedketoneketone of a cyclopentenone
of a cyclopentenone ring observed
ring observed at positions
at positions 1, 2, and1,32, andThe
[39]. 3 [39].
1H- The
1 H-NMR spectrum of C3 exhibited four methyl signals at δ 1.09 (s, C-13), 1.07 (s, C-12),
NMR spectrum of C3 exhibited four methyl signals at δH 1.09 (s, H C-13), 1.07 (s, C-12), 1.04
1.04 (s, C-15),
(s, C-15), and (d,
and 0.96 0.96 6.7J Hz,
J =(d, = 6.7 Hz, which
C-14), C-14), were
which were correlated
correlated in the HSQC in the HSQC spectrum
spectrum with
with the carbon
the carbon signals
signals at δC26.1,
at δC 28.1, 28.1,20.1,
26.1,and
20.1, and
16.2, 16.2, respectively.
respectively. The 13C-NMRThe 13spectrum
C-NMR spectrum
dis-
displayed
played fifteenfifteen
carboncarbon signals,
signals, categorized
categorized as four as four methyls
methyls (δC 20.1,
(δC 26.1, 28.1, 26.1, and
28.1, 20.1,
16.2 as- and
signed
16.2 to C-12,
assigned toC-13,
C-12,C-15,
C-13,and C-14,
C-15, andrespectively), four methylenes
C-14, respectively), (δC 43.5, 35.9,
four methylenes 30.6, 35.9,
(δC 43.5,
and 27.6
30.6, and assigned to C-4,toC-6,
27.6 assigned C-4,C-9, and
C-6, C-8,
C-9, respectively),
and three methines
C-8, respectively), (δC 125.9, (δ
three methines 43.4,
C 125.9,
and 36.5 assigned to C-2, C-7, and C-10, respectively), and four quaternary
43.4, and 36.5 assigned to C-2, C-7, and C-10, respectively), and four quaternary carbon carbon atoms
at δC 202.5
atoms at δC(C-3),
202.5179.8 (C-1),
(C-3), 73.4(C-1),
179.8 (C-11),73.4
and(C-11),
41.9 (C-5).
andThese data, along
41.9 (C-5). These with additional
data, along with
1 1
additional information provided by H- H spin interactions between the coupled protons
for H-6/H-7, H-7/H-8, H-8/H-9, and H-9/H-10, were observed in the COSY spectrum
(Table 1 and Figure S11) establishing a heptocyclic moiety of the sesquiterpene skeleton.
The HMBC correlations (Figures 2 and S12) between the protons at δH 1.07 (s, H-12) and 1.09
(s, H-13) with the carbon signals at δC 73.4 (C-11) and 43.4 (C-7) indicated the presence of a
 Separations 2022, 9, x FOR PEER REVIEW 6 of 18

Separations 2022, 9, 447 6 of 18

information provided by 1H-1H spin interactions between the coupled protons for H-6/H-
7, H-7/H-8, H-8/H-9, and H-9/H-10, were observed in the COSY spectrum (Table 1 and
Figure S11) establishing a heptocyclic moiety of the sesquiterpene skeleton. The HMBC
hydroxyisopropyl group attached to C-7. In addition, the presence of HMBC correlations of
correlations (Figures 2 and S12) between the protons at δH 1.07 (s, H-12) and 1.09 (s, H-13)
H-2/C-5 and H-2/C-9 and H-4/C-1, H-4/C-2, H-4/C-3, H-4/C-5, and H-4/C-6 confirmed
with the carbon signals at δC 73.4 (C-11) and 43.4 (C-7) indicated the presence of a hydrox-
the presence of a cyclopentanone ring system. From the aforementioned data, compound
yisopropyl group attached to C-7. In addition, the presence of HMBC correlations of H-
C3 was identified as a new natural compound named erinaceosin (Figure 1).
2/C-5 and H-2/C-9 and H-4/C-1, H-4/C-2, H-4/C-3, H-4/C-5, and H-4/C-6 confirmed the
presence of a cyclopentanone ring system. From the aforementioned data, compound C3
Table 1. NMR spectral data of compound C3 in CD3 OD (500 MHz for 1 H- and 125 MHz for 13 C-NMR).
was identified as a new natural compound named erinaceosin (Figure 1).
HSQC HMBC (H→C) COSY
Table 1. NMR spectral data of compound
2
C3 in CD3OD
3
(500 MHz for 41H- and 125 MHz
1
for 13C-
Type Hz)
δH (J inNMR). δC JCH JCH JCH H-1 H
1 C 179.8
HSQC HMBC (H→C) COSY
2 CH 5.76, s 125.9 C-5 C-9 4
Type δH (J in Hz) δC J CH
2 3J CH J CH 1H-1H

31 C=O
C 202.5
179.8
42 CH
CH2 2.27,s m; 2.21, m, overlapped
5.76, 43.5
125.9 C-3, C-5 C-5 C-1, C-2, C-6 C-9
53 C=O
C 202.5
41.9
64 CH
CH22 2.27,
1.23,m;m;2.21,
1.88,m,
m overlapped 43.5
35.9 C-3,C-5
C-5 C-1, C-2, C-6 C-11
C-1, C-8, C-10, C-13 H-7
75 CHC 1.36, m 41.9
43.4 C-6, C-11 C-9 C-4 H-6, H-8
6 CH2 1.23, m; 1.88, m 35.9 C-5 C-1, C-8, C-11 C-10, C-13 H-7
8 CH2 1.67, m 27.6 C-9 C-11 C-1 H-7, H-9
7 CH 1.36, m 43.4 C-6, C-11 C-9 C-4 H-6, H-8
9 CH2 2.21, m, overlapped; 2.50, m 30.6 C-1, C-2, C-5, C-6 H-10, H-8
8 CH2 1.67, m 27.6 C-9 C-11 C-1 H-7, H-9
109 CH 2
CH 2.22,m,overlapped
2.21, overlapped; 2.50, m 36.6
30.6 C-1 C-1, C-2, C-5 C-2,C-3,
C-5,C-7
C-6 H-14 H-8
H-10,
11
10 C
CH 2.22, overlapped 73.4
36.6 C-1 C-2, C-5 C-3, C-7 H-14
11
12 CHC3 1.07, s 73.4
26.1 C-11 C-6, C-8
12
13 CH33
CH 1.07,
1.09,s s 26.1
28.1 C-11
C-11 C-12 C-6, C-8
13
14 CH3
CH 1.09,
0.96,s d (6.7) 28.1
16.2 C-11
C-10 C-12 C-5 H-10
3
14 CH3 0.96, d (6.7) 16.2 C-10 C-5 H-10
15 CH3 1.04, s 20.1 C-5 C-1, C-4, C-6
15 CH3 1.04, s 20.1 C-5 C-1, C-4, C-6

Figure 2.
Figure 2. (a)(a)
Selected COSY
Selected correlations
COSY and and
correlations (b) HMBC correlations
(b) HMBC of the new
correlations compounds
of the (C3 and
new compounds
C5).
(C3 and C5).

3.1.3. Identification of Compound C4

The HR-ESI-MS of compound C4 (Figure S21) indicated a molecular formula of
C11 H16 O3 based on the ion peak at m/z 197.1156 [M + H]+ , 100%; 219.0973 [M + Na] + ,
70%. The detailed study of MS, 1D, and 2D NMR data (Table S2 and Figures S14–S20)
identified the structure of C4 as loliolide, which was further confirmed by comparison
Separations 2022, 9, 447 7 of 18

of its spectral data with those reported in the literature. This compound was reported
from genus Echinops for the first time and previously isolated from Codium tomentosum and
Xanthium spinosum [40,41].

3.1.4. Identification of Compound C5

Compound C5 displayed an m/z of 575.1975 [M + 3K] + (100%, cal. 575.1216) in
the positive mode of the HR-ESI-MS spectrum (Figure S29) coincident with a molecular
formula of C26 H34 O7 K3 . One- and two-dimensional NMR spectral data of C5 (Table 2 and
Figures S22–S27) showed the possible presence of an abscisic alcohol moiety [42–45], which
was confirmed by the presence of four tertiary methyl singlets at δH 1.85 (H3 -12; δC 20.2),
1.95 (H3 -13; δC 21.8), 0.98 (H3 -14; δC 24.1), and 0.94 (H3 -15; δC 25.2); two trans-coupled
olefinic proton doublets at δH 7.66 (J = 16.1 Hz, H-8; δC 129.9) and 6.13 (J = 16.1 Hz, H-7;
δC 138.2); and two proton signals at δH 5.67 (1H, br.s, H-10; δC 120.4) and 5.85 (1H, s,
H-3; δC 128.0). The latter two double bonds, 2(3) and 9(10) , bear two methyl substituents
(i.e., H3 -13 and H3 -12) attached to C-2 (δC 152.2) and C-9 (δC 131.0), respectively. The
presence of a hydroxy methylene group was assigned based on the proton multiplet at
δH 3.45 (H-11; δC 62.6). The triene system (∆2(3) , ∆7(8) , and ∆9(10) ) is connected to a carbonyl
carbon at δC 203.0 (C-4), and the connection was confirmed by the HMBC spectrum.
This was evident from the HMBC correlations of the two methylene proton signals at
δH 2.11 and 2.45 (Ha -5 and Hb -5) with the ketonic group at C-4. Other significant HMBC
correlations, including Ha -5/C-1, H-14/C-1, H-15/C-1, H-13/C-2, H-12/C-8, H-3/C-13,
H-3/C-1, H-7/C-1, H-8/C-1, and H-11/C-8, were also used to confirm the presence of an
abscisic alcohol moiety (Table 2 and Figure S27).
In addition to the abscisic alcohol moiety, a set of aromatic ABX systems were revealed
from the doublet signal at δH 6.70 (d, J = 7.8 Hz, H-50 ), a broad doublet at δH 6.45 (br.d,
J = 7.8 Hz, H-60 ), and a broad singlet at δH 6.49 (1H, br.s, H-20 ), which are directly correlated
to carbon signals at δC 116.2, 123.2, and 113.7, respectively (HSQC). The singlet proton
signal at δH 3.64 (3H, s) correlated with the carbon at δC 56.6 (HSQC) and was assigned
to a m-methoxy substituent at C-30 of the aromatic ring. A third moiety formed from a
3-hydroxy-2-methylpropanoic acid chain (HMPA) was attached to C-10 of the aromatic
ring and esterified the abscisic alcohol moiety at C-11. The HMPA moiety was deduced
from the presence of a terminal hydroxy methylene group revealed from a proton signal
that appeared as a doublet of doublet (J = 8.8, 4.5 Hz; H-100 ) overlapped with the proton
multiplet of H2 -11 of the abscisic alcohol moiety at δH 3.45 (4H). This proton signal is directly
correlated with the carbon at δC 62.6 (i.e., coincident signals of C-100 /C-11). Additionally,
the NMR data revealed the presence of two proton multiplets of a methylene group of
H2 -70 (δH 2.49 and 2.65; δC 36.5), which is correlated in the HMBC spectrum with C-8, C-10 ,
C-20 , and C-60 , confirming its attachment to the aromatic ring. Finally, the proton signal
at δH 1.84 (1H, m, H-80 , δC 44.5), forming the branching point of the side chain formed by
C70 -C-80 -C-100 , showed an HMBC correlation with the ester carbonyl group at δC 169.4 (C-90 ).
The above-mentioned data suggested a 4-(3-hydroxypropyl)-2-methoxyphenol moiety that
is closely related to the previously published data of 5-(3-hydroxypropyl)-2-methoxyphenol
derivative [46]. The analysis of the COSY and HMBC spectra of compound C5 (Table 2)
established the connections of the assigned protons and carbons. From the aforementioned
discussion, the structure of C5 was confirmed to be a new derivative composed of an
abscisic alcohol moiety esterified with a modified phenylpropane carboxylic acid moiety
and was named erinaceol, which is recorded herein for the first time from nature.

3.1.5. Identification of Compound C6

Compound C6 was identified as (E)-p-coumaric acid from the 1 H-NMR and APT
spectra (Table S3 and Figures S30 and S31) and by co-chromatography with an authentic
sample of (E)-p-coumaric acid, which was isolated for the first time from E. erinaceus [47].
Separations 2022, 9, 447 8 of 18

3.1.6. Identification of Compound C7

The spectral analysis of C7 (Table S4 and Figures S32–S37) confirmed its structure as
5,7,30 ,50 -tetrahydroxy flavanone [48], and it is worth mentioning that this is the first report
of C7 from Echinops spp.

Table 2. NMR spectral data of compound C5 in CD3 OD (500 MHz for 1 H- and 125 MHz
for 13 C-NMR).

HSQC HMBC (H→C) COSY

C/H# 2J 3J 4J 1 H-1 H
Type δH (J in Hz) δC CH CH CH

Abscisic alcohol moiety

1 C 82.1
2 C 152.2
3 CH 5.85, 1H, s 128.0 C-1, C-13
4 C 203.0
5 CH2 2.11, m; 2.45, m 51.1 C-4, C-6 C-1, C-14, C-15
6 C 43.4
7 CH 6.13, d (16.1) 138.2 C-1, C-8 C-2, C-9 H-7
8 CH 7.66, d (16.1) 129.9 C-7 C-1, C-10, C-12 C-2 H-8
9 C 131.0
10 CH 5.67, br.s 120.4 C-9 C-12
11 CH2 3.45, m a 62.6 b C-8 C-80
12 CH3 1.85, s 20.2 C-8, C-10 C-90
13 CH3 1.95, s 21.7 C-2 C-1, C-3
14 CH3 0.98, s 24.1 C-6 C-1, C-5, C-15
15 CH3 0.94, s 25.1 C-6 C-1, C-5, C-14 C-4
4-(3-Hydroxypropyl)-2-methoxyphenol moiety
10 C 134.4
20 CH 6.49, br. s 113.7 C-30 C-40 , C-70 , C-60
30 C 149.3
40 C 146.4
50 CH 6.70, d (7.8) 116.2 C-40 C-30 , C-10 H-60
60 CH 6.45, br. d (7.9) 123.2 C-40 , C-20 H-50
C-10 ,
70 CH2 2.49, m; 2.65, m 36.5 C-20 , C-60 H-80
C-80
H-70 ,
80 CH 1.84, m 44.5 C-90
H-100
90 C 169.4
100 CH2 3.45, dd (8.8, 4.5) a
62.6 b C-80
O-CH3 3.64, s 56.6 C-30
a, b Similar letters indicate coincident signals.

3.2. Biological Activities of Main Fractions and Isolates from E. erinaceus

3.2.1. In Vitro Cytotoxic Activity
The results of bio-guided cytotoxic activity against HCT-116 and CACO2 cells of the
different extracts showed that the CHCl3 extract showed the highest activity among the
Separations 2022, 9, 447 9 of 18

tested extracts (Table 3). However, it exhibited moderate cytotoxic activity with IC50 of
67.30 ± 4.87 and 81.95 ± 4.63 µg/mL with a selectivity index (SI) > 1 (1.73 and 1.42) against
HCT-116 and CACO2 cells, respectively. Further bio-guided fractionation of the CHCl3
extract revealed that fractions Fr.1, Fr.3, and Fr.4 were the most active among the tested
fractions. Considering Fr.1, it showed the strongest activity with IC50 of 14.93 ± 1.28 and
10.50 ± 0.61 µg/mL and SI of 3.37 and 4.80 against HCT-116 and CACO2 cells, respectively.
Compound C1/C2 showed significant antiproliferative activity with IC50 of 24.95 ± 1.23
and 19.74 ± 1.94 µg/mL and good SI (1.95 and 2.47) against the tested cells, respectively. The
new compound (C3) purified from Fr.3 showed a weak cytotoxic activity (IC50 82.82 ± 3.94
and 99.09 ± 5.84 µg/mL) with good SI (2.15 and 1.80), respectively. Similarly, compound
C5 obtained from Fr.4 showed weak cytotoxicity (IC50 76.70 ± 3.71 and 87.27 ± 4.67 µg/mL,
respectively) and good SI (2.12 and 1.87, respectively) (Table 3).

Table 3. In vitro cytotoxic activity (IC50 , µg/mL), selectivity index (SI) of the different extracts,
fractions, and compounds C1–C7 from E. erinaceus.

IC50 (µg/mL) CC50 (µg/mL) Selectivity Index (SI)

Test Sample
HCT-116 a CACO2 a WI-38 a HCT-116 CACO2
Extract
Total MeOH 165.92 ± 9.82 192.82 ± 12.86 226.14 ± 11.82 1.36 1.17
n-Hex 88.91 ± 5.42 87.93 ± 4.89 110.79 ± 7.43 1.25 1.26
CHCl3 67.30 ± 4.87 ” 81.95 ± 4.63 ” 116.53 ± 9.27 1.73 1.42
EtOAc 170.84 ± 10.29 218.72 ± 11.04 246.41 ± 14.23 1.44 1.13
Re. Aq 323.25 ± 15.83 361.08 ± 18.24 449.72 ± 21.34 1.39 1.25
* CHCl3 Fractions
* Fr.1 14.93 ± 1.28 ” 10.50 ± 0.61 ” 50.36 ± 3.80 3.37 4.80
* Fr.3 30.94 ± 1.78 ” 38.9 ± 1.89 ” 60.62 ± 3.42 1.96 1.56
* Fr.4 24.93 ± 1.29 ” 12.95 ± 0.61 ” 53.24 ± 3.08 2.14 4.11
* Fr.5 83.41 ± 4.03 101.78 ± 4.08 117.64 ± 6.72 1.41 1.16
* Fr.6 54.43 ± 2.19 59.85 ± 2.73 105.31 ± 4.93 1.93 1.76
Compounds
C1/C2 24.95 ± 1.23 ” 19.74 ± 1.94 ” 48.75 ± 3.91 1.95 2.47
C3 82.82 ± 3.94 ” 99.09 ± 5.84 ” 178.02 ± 8.74 2.15 1.80
C4 173.12± 9.74 217.25 ± 8.73 272.93 ± 16.25 1.58 1.26
C5 76.70 ± 3.71 ” 87.27 ± 4.67 ” 162.84 ± 7.08 2.12 1.87
C6 179.81 ± 14.08 425.48 ± 16.71 416.52 ± 18.96 2.32 0.98
C7 219.35 ± 9.76 284.73 ± 14.93 382.53 ± 17.21 1.74 1.34
Vin b 2.35 ± 0.41 2.62 ± 0.44 13.98 ± 1.34 5.95 5.34
a Samples were analyzed in triplicate (n = 3) and expressed as mean ± standard deviation; b
Vin: Vinblastine Sulfate
(Reference standard drug); MeOH = methanol extract; n-Hex = n-hexane extract; CHCl3 = chloroform extract;
EtOAc = ethyl acetate extract; Re.Aq = remaining aqueous extract; * Chloroform fractions; HCT-116 = human
colon cancer cell line; CACO2 = human colorectal intestinal carcinoma cells; WI-38 = human lung fibroblast
normal cells. ” IC50 (µg/mL): 1–10 = very strong, 11–20 = strong, 21–50 = moderate, 51–100 = weak, and above
100 = non-cytotoxic [49].

3.2.2. In Vitro Antimicrobial Activity

The different extracts and fractions of E. erinaceus were tested for their antimicrobial
activity against a panel of pathogenic micro-organisms, including two strains of Gram-
positive bacteria (Bacillus subtilus and methicillin-resistant Staphylococcus aureus, MRSA),
two strains of Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), and two
Separations 2022, 9, 447 10 of 18

fungus and yeast-like micro-organisms (Asperigllus niger and Candida albicans), using the
agar well diffusion assay by measuring the diameter of inhibition zone (DIZ). The results of
the antibacterial properties of the plant extracts (Table 4) demonstrated that the total MeOH
extract had the highest antimicrobial activity against all the tested strains, except against
MRSA. It showed significant antibacterial activity against B. subtilus (27.5 ± 0.7 mm), which
is more active than the reference drug, streptomycin (18 ± 1.41 mm). It also showed a pro-
nounced antifungal activity against C. albicans (26 ± 1.41 mm), which is almost comparable
to the reference drug, clotrimazole (28 ± 2.82 mm). This was followed by the n-hexane and
EtOAc extracts, which showed strong antibacterial and antifungal activities. However, no
antimicrobial activity against MRSA was detected in any of the investigated E. erinaceus
samples. The CHCl3 extract showed good activity against B. subtillis (20.5 ± 1.41 mm),
P. aeruginosa (17.5 ± 1.41 mm), and E. coli (18 ± 1.41 mm) test strains. Fr.3 of the CHCl3
extract showed the highest antimicrobial effect compared to other fractions against the
same bacterial strains as its main extract. However, the CHCl3 extract and its fractions
(Fr.3, Fr.4, and Fr.5) showed no activity against the tested fungal strains, C. albicans and
A. niger. The aqueous extract showed good activity against all strains, except MRSA and
A. niger (Table 4).

Table 4. Antimicrobial activity of the extracts and selected fractions of E. erinaceus against a selected
group of bacterial and fungal pathogens.

Gram-Positive Gram-Negative Fungi and Yeast

Bacterial Isolates B. subtilus (a) MRSA(a) P. aeruginosa (a) E. coli (a) C. albicans (b) A. niger (b)
ATCC6633 ATCC25923 ATCC27953 ATCC25922 NRRLY477 NRRL599
MeOH ext. 27.5 ± 0.7 - 23.5± 0.7 24 ± 1.41 26 ± 1.41 16 ± 1.41
n-hex ext. 22.5 ± 0.7 - 22 ± 2.82 22.25 ± 1.76 22.5 ± 2.82 -
CHCl3 ext. 20.5 ± 1.41 - 17.5 ± 1.41 18 ± 1.41 - -
EtOAc ext. 20.0 ± 1.41 - 22 ± 1.41 21.5 ± 0.7 22 ± 1.41 12.5 ± 0.7
Re. Aq. ext. 18.5 ± 2.12 - 17 ± 1.41 20.5 ± 2.12 18 ± 1.41 -
* Fr.3 17.5± 0.7 - 16 ± 1.41 17.25 ± 2.82 - -
* Fr.4 * 14.5 ± 2.12 - 14 ± 0.71 16 ± 0.7 - -
* Fr.5 * 17 ± 1.41 - 14 ± 1.41 14.5 ± 0.7 - –
Streptomycin a 18 ± 1.41 20 ± 1.41 27 ± 1.41 25 ± 2.82 - -
Clotrimazole b - - - - 28 ± 2.82 26 ± 1.41
Antimicrobial activity of bacterial isolates by agar diffusion method. (a) Grown on nutrient medium agar; (b) on
potato dextrose agar (PDA); diameter of inhibition zone (IZD) measured in mm. Each value is expressed as mean
± SD, pore size 5 mm; -: negative; MeOH = methanol extract; n-hex = n-hexane extract; CHCl3 = chloroform
extract; EtOAc = ethyl acetate extract; and Re. Aq = remaining aqueous extract, tested at a concentration of
200 µg/mL (50 µL/well); * CHCl3 fractions. a : Streptomycin (10 µg/mL) and b : Clotrimazole (15 µg/mL).

3.2.3. Antioxidant Activity

DPPH free radical scavenging activities of Fr.3, Fr.4, and Fr.5 of the CHCl3 fraction
were assessed. Fr.3 exhibited remarkable free radical scavenging activity (55%), whereas
the lowest activity was observed for sample Fr.4 (32%) (Figure 3).

3.3. PASS and ADME Predictions of the Isolated Compounds

The isolated compounds were sketched using the Marvinsketch program and simu-
lated by the Prediction of Activity Spectra for Substances (PASS) and Absorption, Distribu-
tion, Metabolism, and Elimination (ADME) prediction web tools [28–30]. The PASS tool
predicted several biological activities. The isolated compounds (C1/C2, C3–C7) displayed
significant probable activity “Pa” ranges, including antioxidant (0.828–0.297), anticancer
(0.746–0.343), anti-inflammatory (0.717–0.325), antifungal (0.593–0.364), and antibacterial
Separations 2022, 9, 447 11 of 18

(0.455–0.176) activities (Table S5). The ADME prediction results showed that all compounds
showed a great bioavailability score ranging from 0.85 to 0.55 and fulfilled all drug-likeness
rules and synthetic accessibility (1.61–4.14), which showed an explicit synthetic route. Addi-
tionally, all compounds were predicted to be moderate to very soluble in water. In addition,
skin permeation and ADME properties were analyzed by the Swiss-ADME software, as
recorded in Table S5. The BOILED-Egg method [50] showed that compounds C3–C6 have
high GI absorption properties with high predicted diffusion through the BBB and good skin
permeability (log Kp). However, compound C7 showed high predictable GI absorption
and skin permeability properties but may diffuse poorly through the BBB [51]. Compounds
C1–C6 showed no predictable binding to P-gp, except for C7, which may suffer from cellu-
lar efflux. Regarding inhibition of metabolic enzymes, compound C1 may inhibit CYP1A2,
whereas C7 may inhibit CYP3A4, resulting in potential drug–drug interactions and adverse
effects [28]. The findings revealed that five out of the seven compounds fulfilled
Separations 2022, 9, x FOR PEER REVIEW 11 the
of 18oral
drug ability of Lipinski’s rule of five (RO5), while two slightly met the criteria of RO5.

DPPH free radical scavenging activity (%)

100
Scavenging activity %
% Scavenging activity

70
80
55
60
35 32
40
20
0
Fr.5 Fr.4 Fr.3 Ascorbic acid
Samples

Figure 3. DPPH free radical scavenging activity (%). The results are the average of two replicate
Figure 3. DPPH free radical scavenging activity (%). The results are the average of two replicate
experiments, and the error bars show standard deviations.
experiments, and the error bars show standard deviations.
3.3.InPASS
3.4. Silicoand ADME
Docking Predictions
Study of the Isolated
of the Isolated Compounds
Compounds
AThe isolated
docking compounds
study were sketched
was executed using the
for the isolated Marvinsketch
compounds fromprogram and simu-
E. erinaceus against
lated by the Prediction of Activity Spectra for Substances (PASS) and
five explored molecular targets, including DNA gyrase topoisomerase II (PDB ID: 1KZN)Absorption, Distri-[31],
bution, Metabolism, and Elimination (ADME) prediction web tools [28–30]. The PASS tool
enoyl-acyl carrier protein reductase of S. aureus, FabI (PDB ID: 3GNS) [32], and dihydro-
predicted several biological activities. The isolated compounds (C1/C2, C3–C7) displayed
folate reductase (DHFR) (PDB ID: 4M6J) [34] as targets for bacteria, and β-catenin (PDB
significant probable activity “Pa” ranges, including antioxidant (0.828–0.297), anticancer
ID: 7AFW) [33] in addition to human P-glycoprotein (P-gp) (PDB ID: 6C0V) [21] as tar-
(0.746–0.343), anti-inflammatory (0.717–0.325), antifungal (0.593–0.364), and antibacterial
gets for cancer. The results showed that the isolated phenolics and sesquiterpenes were
(0.455–0.176) activities (Table S5). The ADME prediction results showed that all com-
predicted to have remarkable in silico binding affinities against the investigated molecu-
pounds showed a great bioavailability score ranging from 0.85 to 0.55 and fulfilled all
lardrug-likeness
targets. rules and synthetic accessibility (1.61–4.14), which showed an explicit syn-
thetic route. Additionally, all compounds were predicted to be moderate to very soluble
3.4.1. Docking against Antimicrobial Molecular Targets
in water. In addition, skin permeation and ADME properties were analyzed by the Swiss-
Interactions with DNA Gyrase Topoisomerase II
ADME software, as recorded in Table S5. The BOILED-Egg method [50] showed that com-
DNA gyrase
pounds C3–C6 have topoisomerase II is a bacterial
high GI absorption enzyme
properties that predicted
with high regulates the topological
diffusion throughprop-
erties of bacterial
the BBB and good DNA,
skin especially
permeability E. coli.
of (log Kp).Compounds (C1–C6) demonstrated
However, compound C7 showed high moderate–
pre-
weak binding
dictable affinities toward
GI absorption and skinthis protein, asproperties
permeability depicted by
buttheir
may binding free energy
diffuse poorly through(BFE)
values ranging
the BBB from −4.1 to
[51]. Compounds −5.7showed
C1–C6 kcal/mol no (Table S6) compared
predictable binding toto the reference
P-gp, except for ligand,
C7,
which may(CBN,
clorobiocin suffer− from cellular efflux.
7.4 kcal/mol). Regarding
Of these, inhibition
compound C3 of −5.7 kcal/mol)
metabolic
(BFE, enzymes, com-showed
pound C1 interactions
H-bonding may inhibit CYP1A2,
with Ala96 whereas
and theC7crucial
may inhibit
amino CYP3A4, resulting
acid, Gly117 in potential
(Figure 4a), while
drug–drugC5
compound interactions
(BFE, −5.7and adverse effects
kcal/mol) showed[28]. The findings
H-bonding revealedwith
interactions that Ala96
five out
andof Ser121
the
seven compounds fulfilled the oral drug ability of Lipinski’s rule of five (RO5), while two
slightly met the criteria of RO5.

3.4. In Silico Docking Study of the Isolated Compounds

A docking study was executed for the isolated compounds from E. erinaceus against
 Interactions with DNA Gyrase Topoisomerase II
DNA gyrase topoisomerase II is a bacterial enzyme that regulates the topological
properties of bacterial DNA, especially of E. coli. Compounds (C1–C6) demonstrated
moderate–weak binding affinities toward this protein, as depicted by their binding free
energy (BFE) values ranging from −4.1 to −5.7 kcal/mol (Table S6) compared to the refer-
Separations 2022, 9, 447 ence ligand, clorobiocin (CBN, −7.4 kcal/mol). Of these, compound C3 (BFE, −5.7 kcal/mol) 12 of 18

showed H-bonding interactions with Ala96 and the crucial amino acid, Gly117 (Figure
4a), while compound C5 (BFE, −5.7 kcal/mol) showed H-bonding interactions with Ala96
and Ser121
(Figure (Figure
4b) [52]. On4b)
the[52]. Onhand,
other the other hand, compound
compound C7 better
C7 exhibited exhibited
BFEbetter
(−7.7BFE (-7.7
kcal/mol)
kcal/mol)
than CBN,than CBN,
which can which can be by
be explained explained by theofpresence
the presence of several
several binding binding interac-
interactions with the
tions with
amino acidthe amino(Table
residues acid residues (Table S6),
S6), including including
strong strongwith
H-bondings H-bondings with Glu42,
Glu42, Ser121, and the
Ser121,Val120
crucial and theresidue
crucial (Figure
Val120 residue
4c) [52].(Figure 4c) [52].
Therefore, Therefore,
C7 could C7 could
contribute contribute
to the to
antibacterial
the antibacterial
activity of MeOHactivity
and CHClof MeOH
3 and
extracts. CHCl 3 extracts.

Figure 4. Two-dimensional (2D) molecular interactions of (a) Compound C3; (b) Compound C5;
Figure 4. Two-dimensional (2D) molecular interactions of (a) Compound C3; (b) Compound C5;
and (c) Compound C7 with the active site of DNA gyrase topoisomerase II (E. coli) enzyme (PDB
and (c) Compound C7 with the active site of DNA gyrase topoisomerase II (E. coli) enzyme (PDB
ID:1KZN), (dimensions X:21.0176, Y: 30.3575, Z:27.6357), (root mean square deviation) RMSD < 2.
ID:1KZN), (dimensions X:21.0176, Y: 30.3575, Z:27.6357), (root mean square deviation) RMSD < 2.
Interactions with
Interactions with Enoyl-Acyl
Enoyl-Acyl Carrier
CarrierProtein
ProteinReductase
Reductase(FabI)
(FabI)
FabI protein is one of the critical targets for discovering
FabI protein is one of the critical targets for discovering antimicrobial compounds.
antimicrobial It
compounds.
has
It hasbeen
beenfound
foundin in
several bacteria,
several especially
bacteria, in E.incoli
especially and and
E. coli S. aureus. FromFrom
S. aureus. the docking
the dock-
results (Table S6), compounds C4, C-5, and C6 have comparable
ing results (Table S6), compounds C4, C-5, and C6 have comparable BFE valuesBFE values (−5.9, −6.0,
(−5.9,
−and
6.0,−5.8
andkcal/mol, respectively)
−5.8 kcal/mol, to the reference
respectively) FabI inhibitor,
to the reference Triclosan
FabI inhibitor, (BFE, −5.8
Triclosan (BFE,
kcal/mol) [32], while C3 and C7 have greater BFE (−6.3 and −6.9 kcal/mol, respectively). It
− 5.8 kcal/mol) [32], while C3 and C7 have greater BFE (−6.3 and −6.9 kcal/mol, respec-
was observed that the new compound C3 showed strong H-bonding interactions with
tively). It was observed that the new compound C3 showed strong H-bonding interactions
Tyr39, Arg45, Gln64, and Glu72 (Figure 5a), whereas compound C5 (Figure 5b) shared
with Tyr39, Arg45, Gln64, and Glu72 (Figure 5a), whereas compound C5 (Figure 5b) shared
Triclosan in binding with the amino acids Ile20 and Leu196 through the π-alkyl/alky in-
Triclosan in binding with the amino acids Ile20 and Leu196 through the π-alkyl/alky inter-
teraction and formed strong H-bondings with Gly13, Ala21, Ser93, and Ser121 [52]. Fi-
action and formed strong H-bondings with Gly13, Ala21, Ser93, and Ser121 [52]. Finally, C7
nally, C7 was able to interact with several residues, such as Ala15, Ile20, Gly13, and Ser93
was able to interact with several residues, such as Ala15, Ile20, Gly13, and Ser93 (Table S6
(Table S6 and Figure 5c). Consequently, it could be concluded that compounds C3–C5 and
and Figure 5c). Consequently, it could be concluded that compounds C3–C5 and 13
Separations 2022, 9, x FOR PEER REVIEW
C7ofmay
18
be
C7 may be participating in the observed antimicrobial activities of the investigated E. eri-
participating in the observed antimicrobial activities of the investigated E. erinaceus extracts.
naceus extracts.

Figure5.5.Two-dimensional
Figure Two-dimensional (2D)
(2D) molecular
molecularinteractions
interactionsof of
(a)(a)
Compound C3;C3;
Compound (b) (b)
Compound C5; C5;
Compound
and (c) Compound C7 with enoyl-acyl carrier protein reductase of S. aureus (FabI) (PDB ID: 3GNS),
and (c) Compound C7 with enoyl-acyl carrier protein reductase of S. aureus (FabI) (PDB ID: 3GNS),
(dimensions (Å); X:43.7680, Y: 51.7046, Z:49.0095), (root mean square deviation) RMSD < 2.
(dimensions (Å); X:43.7680, Y: 51.7046, Z:49.0095), (root mean square deviation) RMSD < 2.
3.4.2. Docking against Cytotoxic Molecular Targets
Interactions with β-Catenin
In carcinogenesis, the Wnt/β-catenin signal pathway regulates cell proliferation, dif-
ferentiation, and embryonic development [53]. β-catenin is a signaling molecule in the
Separations 2022, 9, 447 Figure 5. Two-dimensional (2D) molecular interactions of (a) Compound C3; (b) Compound13C5; of 18
and (c) Compound C7 with enoyl-acyl carrier protein reductase of S. aureus (FabI) (PDB ID: 3GNS),
(dimensions (Å); X:43.7680, Y: 51.7046, Z:49.0095), (root mean square deviation) RMSD < 2.

3.4.2.
3.4.2.Docking
Docking against
against Cytotoxic Molecular Targets
Cytotoxic Molecular Targets
Interactions with β-Catenin
Interactions with β-Catenin
In
Incarcinogenesis,
carcinogenesis, the the Wnt/β-catenin
Wnt/β-catenin signalsignalpathway
pathwayregulates
regulatescell cell proliferation,
proliferation, dif-
dif-
ferentiation, and embryonic development [53]. β-catenin is a signaling
ferentiation, and embryonic development [53]. β-catenin is a signaling molecule in the molecule in the
Wnt
Wntpathway,
pathway,which
which plays
plays aa central
central role in carcinogenicity
role in carcinogenicity[33,53].[33,53].Any Anyimpairment
impairmentoror
activation of the Wnt pathway leads to cancerous diseases, such
activation of the Wnt pathway leads to cancerous diseases, such as breast, intestine, as breast, intestine,andand
prostate
prostatecancers
cancers[33].
[33]. The
The isolated compounds (C1-C7)
isolated compounds (C1–C7)wereweredocked
dockedinto intothe
theactive
active site
site ofof
β-catenin (PDB ID: 7AFW). They were found to bind to 205–210
β-catenin (PDB ID: 7AFW). They were found to bind to 205–210 and 243–251 amino acid and 243–251 amino acid
residues
residueswith
withBFE between−
BFEbetween 4.2 and
−4.2 −6.0kcal/mol
and −6.0 kcal/mol(Table(TableS6)
S6)[33].
[33].The
Theligand
ligand C5C5 showed
showed
aaBFE of − 5.1 kcal/mol compared to the co-crystallized inhibitor
BFE of −5.1 kcal/mol compared to the co-crystallized inhibitor R9Q (−6.2 kcal/mol) and R9Q ( − 6.2 kcal/mol)
and formed
formed a distinct
a distinct complement
complement to thetobinding
the binding site while
site while orienting
orienting the hydroxyl
the hydroxyl groupgroup
to-
toward the Thr205, and the benzene ring formed π-alkyl interactions
ward the Thr205, and the benzene ring formed π-alkyl interactions with the backbone CH with the backbone CH
ofofLys242 (Figure 6c). This moderate interaction was in full agreement
Lys242 (Figure 6c). This moderate interaction was in full agreement with the obtained with the obtained
cytotoxic
cytotoxic activity
activity of C5 (Table
of C5 (Table 3) 3) and
and suggested
suggested the the Wnt/β-catenin
Wnt/β-catenin signal signalpathway
pathway asasaa
potential
potentialmechanism
mechanism for for its
its cytotoxicity. Regardingcompounds
cytotoxicity. Regarding compoundsC1 C1andandC2, theyshowed
C2,they showed
low
lowBFE values(−
BFEvalues 4.3 and
(−4.3 −4.2kcal/mol),
and −4.2 kcal/mol),although
althoughC1C1sharedsharedthe theco-crystallized
co-crystallized inhibitor
inhibitor
ininbinding
bindingwith
with Asn206.
Asn206. Both compounds (C1
Both compounds (C1andandC2) interactedhydrophobically
C2)interacted hydrophobicallywith with
Lys242 and Pro247 residues through π-alkyl interactions (Figure
Lys242 and Pro247 residues through π-alkyl interactions (Figure 6a,b) [33]. However,6a,b) [33]. However, these
results contradicted
these results the obtained
contradicted high cytotoxic
the obtained activities
high cytotoxic of C1/C2,
activities of C1/C2,which
which suggested
suggested that
they may act through another mechanism.
that they may act through another mechanism.

Figure6.6.Two-dimensional
Figure Two-dimensional (2D) molecular
molecular interactions
interactionsof
of(a)
(a)Compound
CompoundC1 C1and
and(b)(b)C2,
C2,and (c)(c)
and C5C5
withβ-catenin
with β-catenin (PDB
(PDB ID:7AFW),
ID:7AFW), (dimensions
(dimensions(Å);
(Å);X:20.4162,
X:20.4162,Y:Y:21.1198,
21.1198,Z:25.0),
Z:25.0),(root
(rootmean
mean square
square
deviation)RMSD
deviation) RMSD<< 2.
2.

Interactions with Human Dihydrofolate Reductase (DHFR)

The human dihydrofolate reductase (DHFR) protein has a major role in DNA synthesis
in the human and bacterial cell development process. Therefore, it could be considered
a common target for both cytotoxicity and antimicrobial activities [34]. The tested com-
pounds (C1–C7) displayed variable binding affinity to the DHFR protein with BFE values
ranging from −3.8 to −6.3 kcal/mol in comparison with ciprofloxacin (−5.5 kcal/mol) and
methotrexate (−7.1 kcal/mol) (Table S6). Compound C5 showed good BFE (−5.3 kcal/mol),
which is comparable to ciprofloxacin, and showed H-bonding interactions with the amino
acids Gly20, Ser118, Asp145, and Thr146 (Figure 7a), which is in full agreement with the re-
sult of the cytotoxic activity of this compound [33,34]. Notably, compound C7 exhibited the
highest BFE to DHFR enzyme (−6.3 kcal/mol) among the other isolated compounds and
shared ciprofloxacin in binding with Arg77, Ser118, and Ser119 residues (Figure 7b) [34].
Although compounds C1 and C2 showed low binding affinities, they exhibited binding
to Ser119 and Lys55 residues and shared methotrexate in the interaction with Gly20 and
Thr146 residues (Table S6) [34].
 the amino acids Gly20, Ser118, Asp145, and Thr146 (Figure 7a), which is in full agreement
with the result of the cytotoxic activity of this compound [33,34]. Notably, compound C7
exhibited the highest BFE to DHFR enzyme (−6.3 kcal/mol) among the other isolated com-
pounds and shared ciprofloxacin in binding with Arg77, Ser118, and Ser119 residues (Fig-
ure 7b) [34]. Although compounds C1 and C2 showed low binding affinities, they exhib-
Separations 2022, 9, 447 14 of 18
ited binding to Ser119 and Lys55 residues and shared methotrexate in the interaction with
Gly20 and Thr146 residues (Table S6) [34].

Figure 7. Two-dimensional (2D) molecular interactions of (a) compound C5; and (b) compound
Figure 7. Two-dimensional (2D) molecular interactions of (a) compound C5; and (b) compound C7
C7 with the crystal structure of human dihydrofolate reductase (DHFR) bound to NADPH (PDB
with the crystal structure of human dihydrofolate reductase (DHFR) bound to NADPH (PDB
ID:4M6J),(dimensions
ID:4M6J), (dimensions(Å);
(Å);X:20.8191,
X:20.8191,Y:24.1576,
Y:24.1576, Z:27.1117),
Z:27.1117), (root
(root mean
mean square
square deviation)
deviation) RMSD
RMSD << 2.
2.
Interactions with Human P-gp
The P-gp
Interactions protein
with Human is aP-gp
vital protein in MDR to anticancer drugs and other therapeutics
by causing cellular efflux. Therefore, there is a necessity to discover safe MDR inhibitors [21].
The P-gp protein is a vital protein in MDR to anticancer drugs and other therapeutics
Natural products represent a major source of safely used chemotherapeutic drugs [54].
by causing cellular efflux. Therefore, there is a necessity to discover safe MDR inhibitors
The isolated compounds from E. erinaceus were virtually investigated for their binding to
[21]. Natural products represent a major source of safely used chemotherapeutic drugs
P-gp protein. In general, the in silico docking experiments predicted quite strong affinities
[54]. The isolated compounds from E. erinaceus were virtually investigated for their bind-
of the investigated compounds toward P-gp, with BFE values ranging between −6.1
ing
andto−P-gp protein. In
8.4 kcal/mol. general,
Based the in silicodata
on published docking experiments
by Marques et al.,predicted
2021 [21],quite strong
the preferred
affinities of the investigated compounds toward P-gp, with BFE values
binding positions of the P-gp macromolecule are M or H sites. The inhibitors showed ranging between
−6.1 andhydrophobic
strong −8.4 kcal/mol. Based on published
interactions data
at the M site ofby
theMarques et al., 2021
macromolecule, the[21], the preferred
human P-gp (PDB
binding positions of the P-gp macromolecule are M or H sites.
ID: 6C0V), provided mainly by the multiple isoleucine, leucine, phenylalanine, The inhibitors showed
serine,
strong
and tyrosine residues in the binding site (namely Ile340, 731, 735; Leu332, 339; (PDB
hydrophobic interactions at the M site of the macromolecule, the human P-gp Phe72,
ID: 6C0V),
314, 335, provided
336, 728, mainly
732, 759,by the
983;multiple
Ser733, isoleucine, leucine, phenylalanine,
979; and Tyr307, 310). Compound serine,
C3 and
(BFE,
tyrosine
−7.7 kcal/mol) exhibited hydrophobic interactions with Phe335, Leu339, Phe728,335,
residues in the binding site (namely Ile340, 731, 735; Leu332, 339; Phe72, 314, and
336, 728,residues
Phe759 732, 759,(Figure
983; Ser733, 979;was
8a). This andalso
Tyr307, 310).in
observed Compound
the case ofC3 (BFE, −7.7
C5(BFE, −8.4kcal/mol)
kcal/mol),
exhibited hydrophobic
which showed interactions with
several hydrophobic Phe335,with
interactions Leu339, Phe728,
Phe335, andPhe759,
Leu339, Phe759 Ile731,
residuesand
Ile735 (Figure 8b). In some cases, such as in C1, C6, and C7, interactions with the hydroxyl
or carbonyl groups of these ligands with Ser979, Ile736, and Ala729, respectively, were
observed (Figures S42a,g and 8c). On the other hand, the hydroxyl or carbonyl groups
present in other ligands, including C2, C3, C4, and C5, did not contribute much to any
hydrophilic binding affinity (Table S6). Thus, the investigated compounds may act as
potential MDR inhibitors via binding to P-gp protein.
 several hydrophobic interactions with Phe335, Leu339, Phe759, Ile731, and Ile735 (Figure
8b). In some cases, such as in C1, C6, and C7, interactions with the hydroxyl or carbonyl
groups of these ligands with Ser979, Ile736, and Ala729, respectively, were observed (Fig-
ures S42a,g and 8c). On the other hand, the hydroxyl or carbonyl groups present in other
Separations 2022, 9, 447
ligands, including C2, C3, C4, and C5, did not contribute much to any hydrophilic binding
15 of 18
affinity (Table S6). Thus, the investigated compounds may act as potential MDR inhibitors
via binding to P-gp protein.

Figure 8.
Figure 8. Two-dimensional
Two-dimensional (2D)
(2D) molecular
molecular interactions
interactions of
of (a)
(a) Compound
CompoundC3;
C3;(b)
(b)Compound
CompoundC5;
C5;
and (c) Compound C7 with the crystal structure of human P-gp (PDB ID: 6C0V), (dimensions (Å)
and (c) Compound C7 with the crystal structure of human P-gp (PDB ID: 6C0V), (dimensions (Å)
X:20.8191, Y:24.1576, Z:27.1117), (root mean square deviation) RMSD < 2. Width 995, (root mean
X:20.8191, Y:24.1576, Z:27.1117), (root mean square deviation) RMSD < 2. Width 995, (root mean
square deviation) RMSD < 2.
square deviation) RMSD < 2.
4. Conclusions
4. Conclusions
The current
The current study
study includes
includes the the chemical
chemical and and biological
biological evaluations
evaluationsof ofthe
theSaudi
Saudiwild
wild
plant E. erinaceus. Cytotoxic, antioxidant, and antimicrobial activities
plant E. erinaceus. Cytotoxic, antioxidant, and antimicrobial activities were investigated. were investigated.
The phytochemical
The phytochemical investigation
investigation led led to
to the
the isolation
isolation and
and identification
identificationof ofseven
sevenbioactive
bioactive
phytochemicals, including
phytochemicals, including two two unsaturated
unsaturated fattyfatty acid
acidesters,
esters,aapseudoguaiane
pseudoguaianesesquiter-
sesquiter-
pene, aa sesquiterpene
pene, sesquiterpene lactone,
lactone, two
two phenolics,
phenolics, andand an
an abscisic
abscisic alcohol
alcohol derivative.
derivative. Among
Among
the isolated compounds, two compounds were identified for
the isolated compounds, two compounds were identified for the first time from naturethe first time from nature
viz.,
viz., erinaceosin (a pseudoguaiane) and erinaceol (an abscisic alcohol
erinaceosin (a pseudoguaiane) and erinaceol (an abscisic alcohol derivative). In general, derivative). In gen-
eral,results
the the results ofstudy
of this this study demonstrated
demonstrated that that the title
the title plant plant
has has
weak weak to moderate
to moderate cyto-
cytotoxic
toxic activity, however, it showed promising antibacterial, antifungal,
activity, however, it showed promising antibacterial, antifungal, and antioxidant proper- and antioxidant
properties.
ties. The results
The results of the antimicrobial
of the antimicrobial properties
properties of theofplant
the plant extracts
extracts and/orand/or the frac-
the fractions
tions demonstrated
demonstrated that thethat
totalthe total extract
MeOH MeOH had extract had theantimicrobial
the highest highest antimicrobial activity
activity against all
against
the tested allstrains
the tested strains
except except
against against
MRSA. MRSA. Compounds
Compounds C1/C2 showed C1/C2theshowed thecytotoxic
highest highest
cytotoxic
activity activity
against against HCT-116
HCT-116 and CACO and CACO2 cell lines. This research also demonstrated
2 cell lines. This research also demonstrated that the
secondary metabolites isolated from E. from
that the secondary metabolites isolated E. erinaceus
erinaceus have variable
have variable degreesdegrees of binding
of binding affinity
affinity towards the active sites of selected target proteins, including
towards the active sites of selected target proteins, including DNA gyrase topoisomerase DNA gyrase topoi-II,
somerase
FabI, II, FabI,
β-catenin, β-catenin,
DHFR, and DHFR, and P-gp,
P-gp, which may which may to
contribute contribute to their antimicrobial,
their antimicrobial, anticancer,
anticancer,
and and multidrug-resistance
multidrug-resistance inhibition mechanisms.
inhibition mechanisms.

Supplementary Materials:
Supplementary Materials: The
Thefollowing
following supporting
supporting information
information can can be downloaded
be downloaded at:
at: https:
www.mdpi.com/xxx/s1, Figure S1–S37: Spectra of C1–C7; Table
//www.mdpi.com/article/10.3390/separations9120447/s1, FigureS1–S4: NMR
S1–S37: spectral
Spectra data of
of C1–C7; C1,
Tables
C2, C4, NMR
S1–S4: C6, and C7; Table
spectral dataS5:
ofInC1,
silico
C2,physicochemical
C4, C6, and C7;and pharmacokinetics
Table of C1–C7; Table and
S5: In silico physicochemical S6:
Docking results of C1–C7 against PDB ID:1KZN, 3GNS, 7AFW, 4M6J, and 6C0V; Flowchart
pharmacokinetics of C1–C7; Table S6: Docking results of C1–C7 against PDB ID:1KZN, 3GNS, 7AFW, S1: Frac-
tionation and purification of the CHCl3 extract of E. erinaceus; Figure S38–S42: 2D molecular docking
4M6J, and 6C0V; Flowchart S1: Fractionation and purification of the CHCl3 extract of E. erinaceus;
interactions of C1–C7 against PDB ID:1KZN, 3GNS, 7AFW, 4M6J, and 6C0V; Figure S43: Bioavaila-
Figure S38–S42: 2D molecular docking interactions of C1–C7 against PDB ID:1KZN, 3GNS, 7AFW,
bility radar representation of C1–C7; and Figure S44: Predicted BOILED-Egg diagram of C1–C7.
4M6J, and 6C0V; Figure S43: Bioavailability radar representation of C1–C7; and Figure S44: Predicted
Refs. [55,56] is cited in Supplementary Materials.
BOILED-Egg diagram of C1–C7. Refs. [55,56] is cited in Supplementary Materials.
Author Contributions: Conceptualization, E.A.-S., M.M.E.-S. and O.D.E.-G.; methodology, S.H.S.,
F.M.A.B., A.I.F., M.H.A. and N.A.E.; compounds analysis and validation, S.H.S., F.M.A.B. and E.A.-S.;
molecular docking software, S.H.S.; in vitro biological investigations, A.I.F., M.H.A. and N.A.E.;
resources, A.I.F. and M.H.A.; writing-original draft, S.H.S., F.M.A.B. and E.A-S.; review & editing,
all authors; supervision, M.M.E.-S., O.D.E.-G. and E.A.-S. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Separations 2022, 9, 447 16 of 18

Data Availability Statement: The following supporting information can be downloaded at: www.
mdpi.com/xxx/s1, Figures S1–S37: Spectra of C1–C7; Tables S1–S4: NMR spectral data of C1,
C2, C4, C6, and C7; Table S5: In silico physicochemical and pharmacokinetics of C1–C7; Table S6:
Docking results of C1–C7 against PDB ID:1KZN, 3GNS, 7AFW, 4M6J, and 6C0V; Flowchart S1:
Fractionation and purification of the CHCl3 extract of E. erinaceus; Figures S38–S42: 2D molecular
docking interactions of C1–C7 against PDB ID:1KZN, 3GNS, 7AFW, 4M6J, and 6C0V; Figure S43:
Bioavailability radar representation of C1–C7; and Figure S44: Predicted BOILED-Egg diagram
of C1–C7.
Acknowledgments: The authors would like to express a special thanks of gratitude to members of
the Department of Pharmacognosy, Faculty of Pharmacy, Prince Sattam Bin Abdulaziz University, for
their kind help and the use of lab facilities.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Senejoux, F.; Demougeot, C.; Karimov, U.; Muyard, F.; Kerram, P.; Aisa, H.A.; Girard-Thernier, C. Chemical constituents from
Echinops integrifolius. Biochem. Syst. Ecol. 2013, 47, 42–44. [CrossRef]
2. Dong, M.; Cong, B.; Yu, S.-H.; Sauriol, F.; Huo, C.-H.; Shi, Q.-W.; Gu, Y.-C.; Zamir, L.O.; Kiyota, H. Echinopines A and B:
Sesquiterpenoids Possessing an Unprecedented Skeleton from Echinops spinosus. Org. Lett. 2008, 10, 701–704. [CrossRef]
[PubMed]
3. Khadim, E.J.; Abdulrasool, A.A.; Awad, Z.J. Phytochemical Investigation of Alkaloids in the Iraqi Echinops heterophyllus (Com-
positae). Iraqi J. Pharm. Sci. 2014, 23, 26–34. [CrossRef]
4. Kiyekbayeva, L.; Mohamed, N.M.; Yerkebulan, O.; Mohamed, E.I.; Ubaidilla, D.; Nursulu, A.; Assem, M.; Srivedavyasasri,
R.; Ross, S.A. Phytochemical constituents and antioxidant activity of Echinops albicaulis. Nat. Prod. Res. 2018, 32, 1203–1207.
[CrossRef] [PubMed]
5. Lan, H.; Qi-Rong, C.; Rong, L.; Guo-Qiang, L.; Hao, H. A new pentacyclic triterpene, gmeliniin A, from Echinops gmelinii Turcz.
Chin. J. Chem. 2000, 18, 112–114. [CrossRef]
6. Rolnik, A.; Olas, B. The Plants of the Asteraceae Family as Agents in the Protection of Human Health. Int. J. Mol. Sci. 2021, 22,
3009. [CrossRef]
7. Bulut, G.; Haznedaroğlu, M.Z.; Doğan, A.; Koyu, H.; Tuzlacı, E. An ethnobotanical study of medicinal plants in Acipayam
(Denizli-Turkey). J. Herb. Med. 2017, 10, 64–81. [CrossRef]
8. Menut, C.; Lamaty, G.; Weyerstahl, P.; Marschall, H.; Seelmann, I.; Amvam Zollo, P.H. Aromatic plants of tropical Central Africa.
Part XXXI. Tricyclic sesquiterpenes from the root essential oil of Echinops giganteus var. lelyi C. D. Adams. Flavour Fragr. J. 1997,
12, 415–421. [CrossRef]
9. Bitew, H.; Hymete, A. The Genus Echinops: Phytochemistry and Biological Activities: A Review. Front. Pharmacol. 2019, 10, 1234.
[CrossRef]
10. Mustafa, B.; Hajdari, A.; Krasniqi, F.; Hoxha, E.; Ademi, H.; Quave, C.L.; Pieroni, A. Medical ethnobotany of the Albanian Alps in
Kosovo. J. Ethnobiol. Ethnomed. 2012, 8, 6. [CrossRef]
11. Abdallah, H.M.; Ezzat, S.M.; El Dine, R.S.; Abdel-Sattar, E.; Abdel-Naim, A.B. Protective effect of Echinops galalensis against
CCl4-induced injury on the human hepatoma cell line (Huh7). Phytochem. Lett. 2013, 6, 73–78. [CrossRef]
12. Sharma, K.S.; Mishra, S.; Mehta, B.K. Antifertility activity of Echinops echinatus in albino rats. Indian J. Med. Sci. 1988, 42, 23–26.
13. Jin, Q.; Lee, J.W.; Jang, H.; Choi, J.E.; Kim, H.S.; Lee, D.; Hong, J.T.; Lee, M.K.; Hwang, B.Y. Dimeric sesquiterpene and thiophenes
from the roots of Echinops latifolius. Bioorg. Med. Chem. Lett. 2016, 26, 5995–5998. [CrossRef]
14. Alam, M.K.; Ahmed, S.; Anjum, S.; Akram, M.; Shah, S.M.; Wariss, H.M.; Usmanghani, K. Evaluation of antipyretic activity of
some medicinal plants from Cholistan desert Pakistan. Pak. J. Pharm. Sci. 2016, 29, 529–533.
15. Nakano, H.; Ali, A.; Ur Rehman, J.; Mamonov, L.K.; Cantrell, C.L.; Khan, I.A. Toxicity of thiophenes from Echinops transiliensis
(Asteraceae) against Aedes aegypti (Diptera: Culicidae) larvae. Chem. Biodivers. 2014, 11, 1001–1009. [CrossRef]
16. Radulović, N.S.; Denić, M.S. Essential oils from the roots of Echinops bannaticus Rochel ex Schrad. and Echinops sphaerocephalus L.
(Asteraceae): Chemotaxonomic and biosynthetic aspects. Chem. Biodivers. 2013, 10, 658–676. [CrossRef]
17. Sweilam, S.H.; Abdel Bar, F.M.; ElGindi, O.D.; El- Sherei, M.M.; Abdel-Sattar, E.A. Chemical and In Vitro Anti-inflammatory
Assessment of Echinops erinaceus. Trop. J. Nat. Prod. Res. 2021, 5, 715–719.
18. Gillet, J.-P.; Gottesman, M.M. Mechanisms of multidrug resistance in cancer. In Multi-Drug Resistance in Cancer; Springer:
Berlin/Heidelberg, Germany, 2010; pp. 47–76.
19. Wróbel, A.; Eklund, P.; Bobrowska-Hägerstrand, M.; Hägerstrand, H. Lignans and norlignans inhibit multidrug resistance protein
1 (MRP1/ABCC1)-mediated transport. Anticancer Res. 2010, 30, 4423–4428.
20. Luo, L.; Yang, J.; Wang, C.; Wu, J.; Li, Y.; Zhang, X.; Li, H.; Zhang, H.; Zhou, Y.; Lu, A.; et al. Natural products for infectious
microbes and diseases: An overview of sources, compounds, and chemical diversities. Sci. China Life Sci. 2022, 65, 1123–1145.
[CrossRef]
Separations 2022, 9, 447 17 of 18

21. Marques, S.M.; Šupolíková, L.; Molčanová, L.; Šmejkal, K.; Bednar, D.; Slaninová, I. Screening of Natural Compounds as
P-Glycoprotein Inhibitors against Multidrug Resistance. Biomedicines 2021, 9, 357. [CrossRef]
22. Bhabha, G.; Ekiert, D.C.; Jennewein, M.; Zmasek, C.M.; Tuttle, L.M.; Kroon, G.; Dyson, H.J.; Godzik, A.; Wilson, I.A.; Wright, P.E.
Divergent evolution of protein conformational dynamics in dihydrofolate reductase. Nat. Struct. Mol. Biol 2013, 20, 1243–1249.
[CrossRef] [PubMed]
23. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J.
Immunol. Methods 1983, 65, 55–63. [CrossRef] [PubMed]
24. Abd-El-Aziz, A.S.; El-Ghezlani, E.G.; Elaasser, M.M.; Afifi, T.H.; Okasha, R.M. First example of cationic cyclopentadienyliron
based chromene complexes and polymers: Synthesis, characterization, and biological applications. J. Inorg. Organomet. Polym.
Mater. 2020, 30, 131–146. [CrossRef]
25. Gomha, S.M.; Riyadh, S.M.; Mahmmoud, E.A.; Elaasser, M.M. Synthesis and Anticancer Activities of Thiazoles, 1,3-Thiazines,
and Thiazolidine Using Chitosan-Grafted-Poly(vinylpyridine) as Basic Catalyst. Heterocycles 2015, 91, 1227–1243.
26. Mishra, V.; Prasad, D.N. Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics. Int. J.
Food Microbiol. 2005, 103, 109–115. [CrossRef]
27. Burits, M.; Bucar, F. Antioxidant activity of Nigella sativa essential oil. Phytother. Res. 2000, 14, 323–328. [CrossRef]
28. Foudah, A.I.; Alqarni, M.H.; Alam, A.; Salkini, M.A.; Ross, S.A.; Yusufoglu, H.S. Phytochemical Screening, In Vitro and In Silico
Studies of Volatile Compounds from Petroselinum crispum (Mill) Leaves Grown in Saudi Arabia. Molecules 2022, 27, 934. [CrossRef]
29. Filimonov, D.A.; Lagunin, A.A.; Gloriozova, T.A.; Rudik, A.V.; Druzhilovskii, D.S.; Pogodin, P.V.; Poroikov, V.V. Prediction of
the Biological Activity Spectra of Organic Compounds Using the Pass Online Web Resource. Chem. Heterocycl. Compd. 2014, 50,
444–457. [CrossRef]
30. Daina, A.; Michielin, O.; Zoete, V. SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal
chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717. [CrossRef]
31. Lafitte, D.; Lamour, V.; Tsvetkov, P.O.; Makarov, A.A.; Klich, M.; Deprez, P.; Moras, D.; Briand, C.; Gilli, R. DNA gyrase interaction
with coumarin-based inhibitors: The role of the hydroxybenzoate isopentenyl moiety and the 50 -methyl group of the noviose.
Biochemistry 2002, 41, 7217–7223. [CrossRef]
32. Priyadarshi, A.; Kim, E.E.; Hwang, K.Y. Structural insights into Staphylococcus aureus enoyl-ACP reductase (FabI), in complex
with NADP and triclosan. Proteins 2010, 78, 480–486. [CrossRef]
33. Kessler, D.; Mayer, M.; Zahn, S.K.; Zeeb, M.; Wöhrle, S.; Bergner, A.; Bruchhaus, J.; Ciftci, T.; Dahmann, G.; Dettling, M.; et al.
Getting a Grip on the Undrugged: Targeting β-Catenin with Fragment-Based Methods. ChemMedChem 2021, 16, 1420–1424.
[CrossRef]
34. Khatun, M.; Muhit, M.; Hossain, M.J.; Al-Mansur, M.; Rahman, S.M. Isolation of phytochemical constituents from Stevia
rebaudiana (Bert.) and evaluation of their anticancer, antimicrobial and antioxidant properties via in vitro and in silico approaches.
Heliyon 2021, 7, e08475. [CrossRef]
35. Chira, N.; Nicolescu, A.; Raluca, S.; Rosca, S. Fatty Acid Composition of Vegetable Oils Determined from 13 C-NMR Spectra. Rev.
Chim. (Bucharest) 2016, 67, 1257–1263.
36. Di Pietro, M.E.; Mannu, A.; Mele, A. NMR Determination of Free Fatty Acids in Vegetable Oils. Processes 2020, 8, 410. [CrossRef]
37. Hymete, A.; Rohloff, J.; Kjøsen, H.; Iversen, T.-H. Acetylenic thiophenes from the roots of Echinops ellenbeckii from Ethiopia. Nat.
Prod. Res. 2005, 19, 755–761. [CrossRef]
38. Lao, A.; Fujimoto, Y.; Tatsuno, T. Studies on the Constituents of Artemisia argyi LEVL et VANT. Chem. Pharm. Bull. 1984, 32,
723–727. [CrossRef]
39. Atta-Ur-Rahman; Ahmad, V.U. 13 C-NMR of Natural Prodacts: Volume 1 Monoterpenes and Sesquiterpenes, 1st ed.; Springer: Boston,
MA, USA, 1992; Volume 1, pp. X–966.
40. Yuan, Z.; Zheng, X.; Zhao, Y.; Liu, Y.; Zhou, S.; Wei, C.; Hu, Y.; Shao, H. Phytotoxic Compounds Isolated from Leaves of the
Invasive Weed Xanthium spinosum. Molecules 2018, 23, 2840. [CrossRef]
41. Silva, J.; Alves, C.; Martins, A.; Susano, P.; Simões, M.; Guedes, M.; Rehfeldt, S.; Pinteus, S.; Gaspar, H.; Rodrigues, A.; et al.
Loliolide, a New Therapeutic Option for Neurological Diseases? In Vitro Neuroprotective and Anti-Inflammatory Activities of a
Monoterpenoid Lactone Isolated from Codium tomentosum. Int. J. Mol. Sci. 2021, 22, 1888. [CrossRef]
42. Milborrow, B.V. The conformation of abscisic acid by NMR. and a revision of the proposed mechanism for cyclization during its
biosynthesis. Biochem. J. 1984, 220, 325–332. [CrossRef] [PubMed]
43. Ferreres, F.; Andrade, P.; Tomás-Barberán, F.A. Natural Occurrence of Abscisic Acid in Heather Honey and Floral Nectar. J. Agric.
Food Chem. 1996, 44, 2053–2056. [CrossRef]
44. Frackenpohl, J.; Grill, E.; Bojack, G.; Baltz, R.; Busch, M.; Dittgen, J.; Franke, J.; Freigang, J.; Gonzalez, S.; Heinemann, I.;
et al. Insights into the in Vitro and in Vivo SAR of Abscisic Acid—Exploring Unprecedented Variations of the Side Chain via
Cross-Coupling-Mediated Syntheses. Eur. J. Org. Chem. 2018, 2018, 1403–1415. [CrossRef]
45. Jo, M.S.; Lee, S.; Yu, J.S.; Baek, S.C.; Cho, Y.-C.; Kim, K.H. Megastigmane Derivatives from the Cladodes of Opuntia humifusa and
Their Nitric Oxide Inhibitory Activities in Macrophages. J. Nat. Prod. 2020, 83, 684–692. [CrossRef] [PubMed]
46. Ng, V.A.; Agoo, E.M.; Shen, C.-C.; Ragasa, C. Chemical Constituents of Cycas aenigma. J. Appl. Pharm. Sci 2015, 5, 32–36. [CrossRef]
47. Sytar, O.; Hemmerich, I.; Zivcak, M.; Rauh, C.; Brestic, M. Comparative analysis of bioactive phenolic compounds composition
from 26 medicinal plants. Saudi J. Biol. Sci. 2016, 25, 631–641. [CrossRef]
Separations 2022, 9, 447 18 of 18

48. Nessa, F.; Ismail, Z.; Mohamed, N. Xanthine oxidase inhibitory activities of extracts and flavonoids of the leaves of Blumea
balsamifera. Pharm. Biol. 2010, 48, 1405–1412. [CrossRef]
49. Ayyad, S.E.; Abdel-Lateff, A.; Alarif, W.M.; Patacchioli, F.R.; Badria, F.A.; Ezmirly, S.T. In vitro and in vivo study of cucurbitacins-
type triterpene glucoside from Citrullus colocynthis growing in Saudi Arabia against hepatocellular carcinoma. Environ. Toxicol.
Pharmacol. 2012, 33, 245–251. [CrossRef]
50. Daina, A.; Zoete, V. A BOILED-Egg To Predict Gastrointestinal Absorption and Brain Penetration of Small Molecules. ChemMed-
Chem 2016, 11, 1117–1121. [CrossRef]
51. Bojarska, J.; Remko, M.; Breza, M.; Madura, I.D.; Kaczmarek, K.; Zabrocki, J.; Wolf, W.M. A Supramolecular Approach to
Structure-Based Design with A Focus on Synthons Hierarchy in Ornithine-Derived Ligands: Review, Synthesis, Experimental
and in Silico Studies. Molecules 2020, 25, 1135. [CrossRef]
52. Kandsi, F.; Elbouzidi, A.; Lafdil, F.Z.; Meskali, N.; Azghar, A.; Addi, M.; Hano, C.; Maleb, A.; Gseyra, N. Antibacterial and
Antioxidant Activity of Dysphania ambrosioides (L.) Mosyakin and Clemants Essential Oils: Experimental and Computational
Approaches. Antibiotics 2022, 11, 482. [CrossRef]
53. He, S.; Tang, S. WNT/β-catenin signaling in the development of liver cancers. Biomed. Pharmacother. 2020, 132, 110851. [CrossRef]
54. Li, W.; Zhang, H.; Assaraf, Y.G.; Zhao, K.; Xu, X.; Xie, J.; Yang, D.H.; Chen, Z.S. Overcoming ABC transporter-mediated multidrug
resistance: Molecular mechanisms and novel therapeutic drug strategies. Drug Resist. Updates 2016, 27, 14–29. [CrossRef]
55. Guzel, A.; Aksit, H.; Elmastas, M.; Erenler, R. Bioassay-guided isolation and identification of antioxidant flavonoids from
Cyclotrichium origanifolium (Labill.) Manden and Scheng. Pharmacogn. Mag. 2017, 13, 316–320. [CrossRef]
56. Hussein, N.; Amen, Y.; Abdel Bar, F.; Halim, A.; Saad, H.-E. Antioxidants and α-Glucosidase Inhibitors from Lactuca serriola L.
Rec. Nat. Prod. 2020, 14, 410–415. [CrossRef]