molecules
Article
In Silico Studies of Four Compounds of Cecropia obtusifolia
against Malaria Parasite
Carlos Alberto Lobato-Tapia 1 , Yolotl Moreno-Hernández 2
Citation: Lobato-Tapia, C.A.;
Moreno-Hernández, Y.; Olivo-Vidal,
Z.E. In Silico Studies of Four
Compounds of Cecropia obtusifolia
against Malaria Parasite. Molecules
2023, 28, 6912. https://doi.org/
10.3390/molecules28196912
Academic Editor: Abdelwahab Omri
Received: 10 August 2023
Revised: 4 September 2023
Accepted: 5 September 2023
Published: 3 October 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/).
Molecules 2023, 28, 6912. https://doi.org/10.3390/molecules28196912
and Zendy Evelyn Olivo-Vidal 2, *
1 Departamento de Ingeniería en Biotecnología, Universidad Politécnica Metropolitana de Puebla,
Popocatépetl s/n, Reserva Territorial Atlixcáyotl, Tres Cerritos, Puebla 72480, Mexico
2 Departamento de Salud, El Colegio de la Frontera Sur Unidad Villahermosa, Carretrea Federal
Villa-Hermosa-Reforma Km 15.5, Ra. Guineo Segunda Sección, C.P., Villahermosa 86280, Mexico;
yolotl.moreno@estudianteposgrado.ecosur.mx
* Correspondence: ozendy@ecosur.mx
Abstract: Malaria is a disease that affects many people in the world. In Mexico, malaria remains an
active disease in certain regions, particularly in the states of Chiapas and Chihuahua. While anti-
malarial effects have been attributed to some species of Cecropia in various countries, no such studies
have been conducted in Mexico. Therefore, the objective of this study was to evaluate the in silico
antimalarial activity of some active compounds identified according to the literature in the species
of Cecropia obtusifolia, belonging to the Cecropiaceae family, such as ursolic acid, α-amyrin, chrysin,
and isoorientin. These compounds were evaluated with specific molecular docking and molecular
dynamics (MD) studies using three different malarial targets with the PDB codes 1CET, 2BL9, and
4ZL4 as well as the prediction of their pharmacokinetic (Pk) properties. Docking analysis revealed the
following best binding energies (kcal/mol): isoorientin–1CET (−9.1), isoorientin–2BL9 (−8.8), and
chrysin–4ZL4 (−9.6). MD simulation validated the stability of the complexes. Pharmacokinetics anal-
ysis suggested that the compounds would generally perform well if administered. Therefore, these
results suggest that these compounds may be used as potential drugs for the treatment of malaria.
Keywords: antimalarial; plasmodium; molecular docking; molecular dynamics; Cecropia obtusifolia;
pharmacokinetics
1. Introduction
Malaria is a disease that affects many people worldwide but mainly newborns, children
under five years of age, pregnant women, travelers, and people with a compromised
immune system are at risk of developing a major infection [1]. It is a disease caused by
the bite of a female mosquito of the genus Anopheles spp. which requires feeding for
reproduction; the infection of this disease causes symptoms such as fevers, headache, and
chills, symptoms that can be implicated in fatigue, confusion, seizures, shortness of breath,
anemia, coma, and death [1,2].
In 2021, half of the world’s population was at risk of malaria and during the same
year, there were 247 million cases and 619,000 deaths mainly in the African region where
the incidence is 232.8 (cases per 1000 people at risk) [2]. P. vivax is the second most studied
species, only below P. falciparum due to the severity of the disease it causes and its wide
distribution; in Central America alone, P. vivax is the etiologic agent of 95% of diagnosed
malaria cases and in the region of the Americas the incidence is 4.6. Some standardized
treatments to combat this disease have been generating resistance due to bad practice or
continuous use. Many of the current drugs have been developed by the use of plants in
a traditional way and by studies of some compounds present in the species with some
activity, such as some flavonoids of plant species that have been described with antimalarial
potential, although many more are unknown [3–5]. In Mexico, malaria remains endemic
in states like Chiapas and Chihuahua where several communities are affected year after
https://www.mdpi.com/journal/molecules
 activity, such as some flavonoids of plant species that have been described with
antimalarial potential, although many more are unknown [3–5]. In Mexico, malaria
Molecules 2023, 28, 6912 remains endemic in states like Chiapas and Chihuahua where several communities 2 ofare
14
affected year after year [6]. Cecropia obtusifolia is a perennial tree distributed from Mexico
to Brazil, that has been extensively studied for its pharmacological properties and use in
the treatment
year [6]. Cecropia of obtusifolia
chronic diseases [7–9]. tree
is a perennial These properties
distributed fromhave been to
Mexico attributed
Brazil, that to has
the
presence
been of activestudied
extensively compounds for its such as flavonoids, proantho-cyanins,
pharmacological properties and useterpenoids, steroids,
in the treatment of
chlorogenic
chronic acid,[7–9].
diseases caffeicThese
acid, properties
storminoushave acid,been
andattributed
isoorientinto[10–12], among
the presence of others,
active
primarily found
compounds such in the leaves, proantho-cyanins,
as flavonoids, bark, stem, flower,terpenoids,
bud, and steroids,
root of these plants acid,
chlorogenic [13].
Recently,
caffeic thisstorminous
acid, species hasacid, beenand attributed to moderate
isoorientin [10–12],antiplasmodial
among others, effects, which
primarily found couldin
promote
the leaves,itsbark,
use stem,
for the treatment
flower, bud, and of malaria [14], plants
root of these an infectious diseasethis
[13]. Recently, caused
speciesby has
the
been attributed
Plasmodium spp.to moderate
parasite andantiplasmodial
transmitted to humanseffects, which
by thecould promote
Anopheles its use for the
spp. vector.
treatment of malaria
To date, [14], anhave
no studies infectious disease
evaluated thecaused Plasmodium activity
by theantimalarial
potential spp. parasite
of someand
transmitted
compounds to of humans
Cecropia by the Anopheles
obtusifolia. On thespp.
othervector.
hand, some of the most targeted molecular
To date, no
or therapeutic studies
sites in thehavesearchevaluated the potential
for antimalarial drugs antimalarial activity
are the proteins of some
2BL9, 4ZL4,com- and
pounds
1CET. The of Cecropia
protein obtusifolia. On the other reductase
2BL9, or dihydrofolate hand, some of the of
protein most targeted molecular
Plasmodium vivax, is a
or therapeutic
crucial target sites
in in thethedevelopment
search for antimalarial
of new drugs are the proteins
antimalarial 2BL9, 4ZL4,
agents [15,16] suchand as
1CET. The protein 2BL9, or dihydrofolate reductase protein
pyrimentamine, a treatment for the elimination of protozoa like plasmodium of Plasmodium vivax, is a crucial or
target in the development
toxoplasmosis [17]. Similarly, of new antimalarial
the protein agents [15,16]
4ZL4 belongs such as protease
to the aspartyl pyrimentamine,
known as a
treatment
plasmepsin forVthe elimination
of P. vivax and of protozoa
it is the site like plasmodium
of action or toxoplasmosis
of synthetically formulated [17]. Similarly,
compounds
the protein malaria
to combat 4ZL4 belongssuch as to WEHI-842
the aspartyl protease known
[12,18–20]. Lastly, astheplasmepsin
protein 1CET V ofisP.associated
vivax and
it is the site of action of synthetically formulated compounds
with the Plasmodium falciparum lactate dehydrogenase cofactor binding site, which to combat malaria such as
is the
WEHI-842 [12,18–20]. Lastly, the protein 1CET is associated with
main binding and action site for chloroquine, one of the most widely used drugs to treat the Plasmodium falciparum
lactate
malariadehydrogenase
[19,21,22]. cofactor binding site, which is the main binding and action site for
chloroquine, one of
Therefore, the main the most widely
objective ofused drugs was
this study to treat malaria the
to evaluate [19,21,22].
potential antimalarial
Therefore, the main objective of this study was
activity of the metabolites identified formerly in C. obtusifolia through to evaluate the potential
in silicoantimalarial
analysis, as
activity of the metabolites identified
well as to assess their pharmacokinetic properties. formerly in C. obtusifolia through in silico analysis, as
well as to assess their pharmacokinetic properties.
2. Results
2. Results
2.1. Compounds
2.1. Compounds of of Cecropia
Cecropia obtusifolia
obtusifolia
Due to
Due to some
some background
background in in the
the use
use of
of flavonoids
flavonoids from
from other
other species
species with
with potential
potential
antimalarial activity, it was decided to work with flavonoids such as chrysin, isoorientin,
antimalarial activity, it was decided to work with flavonoids such as chrysin, isoorientin,
and triterpenes
and triterpenes such
such asas ursolic
ursolic acid
acid and
and α-amyrin
α-amyrin present
present in
in this
this species
species Figure
Figure 1.
1.

Figure 1.
1. Chemical structures of the compounds
compounds identified in C.
C. obtusifolia
obtusifolia and
and evaluated
evaluated against
against
target proteins in malaria.
target proteins in malaria.

2.2. Molecular Docking

Molecular docking analysis was performed to assess the interaction capacity between
the target proteins and the compounds found in C. obtusifolia, using Autodock Vina to
calculate the binding energy. The three target proteins used were 2BL9, 4ZL4, and 1CET,
Molecules 2023, 28, 6912 3 of 14

which play a key role in malaria treatment and have known binding sites in their crystalline
structures (found in the .pdb file) with a 25 Å grid.
The binding energy of all compounds was below −7.7 kcal/mol, as shown in Table 1.
The best complexes between the ligands and target were chrysin–2BL9 (−8.7 kcal/mol),
chrysin–4ZL4 (−9.6 kcal/mol) and isoorientin–1CET (−9.1 kcal/mol). In fact, almost
all the evaluated ligands exhibited higher affinities than the co-crystallized ligands in
the re-docking, except for ursolic acid with 4ZL4 (−7.8 kcal/mol) compared to Wehi
(−8.2 kcal/mol).

Table 1. Binding affinities and ligand–amino acid interactions.

Binding Affinities Amino Acid

Target Ligand
(Kcal/mol) Interactions
α-amyrin −7.9 K55, Y56, V60, Y63, K169, Y192, S220, Y223, F232,
Chrysin −8.7 C14, A15, D53, F57, S117, S120, I121, I173, G175, Y179
2BL9 Isoorientin −8.5 L39, L45, M54, F57, S116, S120, I121, Y125, I173, G175
Ursolic acid −7.8 K55, Y56, V60, Y63, Y192, S220, E221, F232
Pyrimithamine −7.6 I13, C14, A15, L45, D53, M54, Y56, F57, I173, Y179
α-amyrin −8.2 I24, D25, T285, F286, H288, K390, E399, L400, V402
Chrysin −9.6 Y27, D48, Y107, E109, L147, F148, V156
4ZL4 Isoorientin −8.3 A28, G50, S51, C108, E109, D281, G283, T285, H288
Ursolic acid −7.8 D25, I24, E109, T285, F286, V354, K390
Wehi −8.2 Y29, D48, S51, E109, Q151, G283, S284, T285, F286, H288, V354, V357
α-amyrin −7.9 G11, G13, M14, P34, D35, I36, V37, A80, G81, F82, I105
Chrysin −7.8 G11, G13, D35, I36, A80, Y67, F82, G81, I105
1CET Isoorientin −9.1 S12, C13, I36, G11, N35, Y67, A80, G81, T83, E108
Ursolic acid −7.7 F34, N35, I36, V37, Y67, A80, G81, I105, I109
Chloroquine −6.3 G11, F34, D35, A80, G81, F82, I36, Y67, I105

Figure 2 illustrates the binding site and orientation pose of these three complexes. The
interactions formed between these compounds and the target proteins were visualized
using PyMOL 2.5.4 and LigPlot 2.2.5. Chrysin was found to form hydrophobic interactions
with Ile13, Cys14, Ala15, Asp53, Tyr56, Phe57, Ser120, Ile121, Ile173, Gly174, and Gly175
and hydrogen bond with Ser117 (3.03 Å) from the 2BL9 target. Chrysin also exhibited
hydrophobic interactions with Tyr27, Ile46, Tyr107, Cys108, Leu147, Phe148, Gln151, Val156,
and Gly283, along with two hydrogen bonds with Asp48 (2.77 Å) and Glu109 (3.06 Å) from
4ZL4. Isoorientin demonstrated hydrophobic interactions with Gly11, Ser12, Gly13, Met14,
Ile36, Met40, Ala80, Phe82, and Lys104 as well as five hydrogen bonds with Asp35 (3.16 Å),
Gly81 (2.91 Å), Thr83 (3.24 Å), and Glu108 (2.69 Å) from 1CET.
Overall, chrysin showed the strongest binding affinity for both 2BL9 and 4ZL4. Its
interaction with 2BL9 involved residues Cys14, Ala15, Asp53, Phe57, Ile173, and Tyr179
which coincide with pyrimethamine. In contrast, its interaction with 4ZL4 only aligned
with a Wehi-binding residue (Asp48). The high affinity of chrysin for these two receptors
positions it as a promising compound for malaria treatment, particularly in the eradication
of P. vivax. On the other hand, isoorientin demonstrated the highest affinity among the
compounds tested with 1CET.
The validation of molecular docking was performed by re-docking the ligands into
the active target sites. Ligand re-docking was conducted after randomizing the ligand’s
conformation and orientation. RMSD values were manually calculated using PyMOL,
resulting in the following values: 0.635 Å (1CET-Chloroquine), 0.333 Å (4ZL4-WEHI), and
0.639 Å (2BL9-pyrimethamine).
Molecules 2023,28,
Molecules2023, 28,6912
x FOR PEER REVIEW 44 of 14
15

Figure 2.2. Two-

Figure Two-and
andthree-dimensional
three-dimensionalrepresentations
representations of
ofthethehydrogen
hydrogenbonding
bonding and
andhydrophobic
hydrophobic
interaction between ligands
interaction ligands within
within thethebinding
bindingcavity
cavityofofreceptors.
receptors.(a)(a)
Chrysin–2BL9 complex,
Chrysin–2BL9 (b)
complex,
chrysin–4ZL4
(b) chrysin–4ZL4 complex,
complex,andand
(c) (c)
isoorientin–CET complex.
isoorientin–CET complex.

2.3. Molecular
Overall, Dynamics
chrysin showed the strongest binding affinity for both 2BL9 and 4ZL4. Its
interaction with 2BL9 the
After evaluating involved residues Cys14,
protein–ligand Ala15,and
interactions Asp53, Phe57,satisfactory
obtaining Ile173, and results,
Tyr179
which coincide
molecular with (MD)
dynamics pyrimethamine.
analysis wasInconducted
contrast, its
oninteraction
the ligandswith
with4ZL4 only aligned
the highest affin-
with a Wehi-binding residue (Asp48). The high affinity of chrysin for these two
ity energies to their targets to assess the stability of the protein–ligand complexes. MDreceptors
positions itwere
simulations as aperformed
promisingforcompound forcomplexes
50 ns for the malaria with
treatment, particularly
the lowest in the
affinity energy:
eradication of chrysin–4ZL4,
chrysin–2BL9, P. vivax. On the other
and hand, isoorientin demonstrated the highest affinity
isoorientin–1CET.
among the compounds tested with 1CET.
 Molecules 2023, 28, 6912 5 of 14

To analyze the structural stability and fluctuations of the complexes, the trajecto-
ries were analyzed using RMSD (root mean square deviation), RMSF (root mean square
fluctuation), RG (radius of gyration), and SASA (solvent accessible surface area).
The RMSD of the protein backbone was plotted against the 50 ns simulation duration
to assess variations in structural conformation (Figure 3). The chrysin–2BL9 complex
exhibited significant variation (0.3 nm) around 20 ns, but after this time, it achieved a
stable conformation without significant deviations in the RMSD values. The chrysin–4ZL4
complex showed variations in backbone RMSD ranging between 0.4 to 0.7 nm from 10 to
30 ns. The stable conformation was reached between 33 and 50 ns with no considerable
deviations in the values. The isoorientin–1CET complex reached equilibrium after 15 ns and
Molecules 2023, 28, x FOR PEER REVIEWremained stable until 50 ns. The RMSD values for both the 4ZL4–chrysin and 2BL9–chrysin 6 of 15
complexes were slightly higher than those of the proteins bound to their co-crystallized
ligands, while the 1CET–isoorientin complex showed lower values compared to the control.

Figure
Figure 3. MSD
3. MSD and
and RMSFplots
RMSF plotsfor
for50
50ns
ns MD
MD simulation
simulationofof1CET–isoorietin
1CET–isoorietin(a),(a),
4ZL4–chrysin (b), (b),
4ZL4–chrysin
and 2BL9–chrysin (c) complex versus co-crystallized ligand.
and 2BL9–chrysin (c) complex versus co-crystallized ligand.

TheThe RMSF
RMSF plotwas
plot wasused
usedtotoanalyze
analyze the
the flexibility
flexibilityofofeach
eachamino
aminoacid residue
acid in the
residue in the
target protein upon binding to a ligand (Figure 3). In the chrysin–2BL9 complex, the amino
target protein upon binding to a ligand (Figure 3). In the chrysin–2BL9 complex, the amino
acids that interacted with chrysin showed minimal fluctuation values. Although some
acids that interacted with chrysin showed minimal fluctuation values. Although some
residues (30, 31, 63, 74, 81–84, 96, and 141–144) displayed larger fluctuations, these residues
residues
are not (30, 31,to63,
related the 74, 81–84,
binding site 96, andHowever,
pocket. 141–144)in displayed largercomplex,
the chrysin–4ZL4 fluctuations, these
a greater
residues are not related to the binding site pocket. However, in the chrysin–4ZL4
number and larger fluctuations of residues were observed throughout the protein. The complex,
a greater number and larger fluctuations of residues were observed throughout the
protein. The residues interacting with the ligand did not show high fluctuations but
greater variability was observed in other regions. Finally, the isoorientin–CET complex
exhibited minimal fluctuation values, except for residues 169–172, which are not involved
 Molecules 2023, 28, 6912 6 of 14

residues interacting with the ligand did not show high fluctuations but greater variability
was observed in other regions. Finally, the isoorientin–CET complex exhibited minimal
fluctuation values, except for residues 169–172, which are not involved in the binding site
pocket. The fluctuations observed with the evaluated compounds are very similar to those
of the co-crystallized complexes.
RG values of the protein backbone atoms were used to analyze the compactness and
structural flexibility of the complex over simulation time (Figure 4). In the chrysin–2BL9
complex, the RG values decreased until 10 ns and remained relatively stable thereafter.
Similarly, in the chrysin–4ZL4 complex, the RG values decreased after 10 ns with small
fluctuations, albeit greater than in the chrysin–2BL9 complex. However, in the isoorientin–
x FOR PEER REVIEW 1CET complex, an increase in RG value was observed after approximately 7 15
of ns,
15 remaining
stable until the end of the dynamics. When comparing these results with those obtained
from the co-crystallized ligands, it is evident that the compounds from C. obtusifolia tend to
have lower RG values.

Figure 4. Radius of gyration and solvent accessible surface area plots for 50 ns MD simulation of the
1CET–isoorietin, 4ZL4–chrysin, and 2BL9–chrysin complex versus co-crystallized ligand.
Figure 4. Radius of gyration and solvent accessible surface area plots for 50 ns MD simulation of the
1CET–isoorietin, 4ZL4–chrysin, and 2BL9–chrysin
Furthermore, the changescomplex versus area
in the surface co-crystallized
(SASA) forligand.
all complexes were plotted
against time to estimate the variations (Figure 4). In all complexes, a decrease in SASA
Furthermore, the changes
values in the surface
was observed until 20 area (SASA) for
ns. Moreover, bothall
thecomplexes were plotted
1CET–isoorientin and 4ZL4–chrysin
against time to estimate the variations
complexes exhibited a(Figure
greater 4). In all in
decrease complexes, a decrease
SASA compared in SASA
to when the targets were
values was observed until 20 ns. Moreover, both the 1CET–isoorientin and 4ZL4–chrysin
complexes exhibited a greater decrease in SASA compared to when the targets were
bound to their co-crystallized ligands (chloroquine and Wehi, respectively). In contrast,
the 2BL9–chrysin complex showed average SASA values higher than those with the co-
Molecules 2023, 28, 6912 7 of 14

bound to their co-crystallized ligands (chloroquine and Wehi, respectively). In contrast,

the 2BL9–chrysin complex showed average SASA values higher than those with the co-
crystallized ligand (pyrimithamine).

2.4. Pharmacokinetic Properties

The ADMET properties of four compounds found in C. obtusifolia were assessed using
the ADMETlab 2.0 web server. The main pharmacokinetics parameters are presented in
Table 2 and complete results can be reviewed in the Supplementary Material Table S1.
Isoorientin was the only compound that did not meet the criteria according to Lipinski’s
five rules and was not considered a potential drug. Similarly, isoorientin exhibited a low
LogP value, indicating higher water solubility compared to the other compounds which are
more liposoluble. The results also showed that α-amyrin, chrysin, and ursolic acid have the
potential for excellent intestinal absorption (HIA) and permeability through Caco-2 cells,
as indicated by ADMETlab 2.0 interpretation ranges. However, isoorientin is expected to
have poor absorption and permeability.

Table 2. Main pharmacokinetics parameters of compounds from C. obtusifolia.

Parameter α-Amyrin Chrysin Isoorientin Ursolic Acid

Formula C30 H50 O C15 H10 O4 C21 H20 O11 C30 H48 O3
MW (g/mol−1 ) 426.39 254.06 448.1 456.36
Lipinski Accepted Accepted Rejected Accepted
LogP (log mol/L) 7.603 3.58 0.708 6.453
HIA 0.003 0.012 0.909 0.004
Caco-2 −5.131 −4.874 −6.251 −5.396
PPB (%) 97.63 98.03 90.58 97.44
VD (L/kg) 1.251 0.493 0.834 0.672
BBB 0.056 0.02 0.011 0.265
CL (mL/min/kg) 5.609 5.131 4.067 3.538
T1/2 0.075 0.787 0.823 0.07
hERG 0.042 0.037 0.214 0.007
H-HT 0.156 0.079 0.133 0.435
Carcinogencity 0.012 0.317 0.037 0.085

The distribution parameter was evaluated for plasma protein binding (PPB), volume
distribution (VD), and the blood–brain barrier (BBB). All the compounds exhibited very
high affinity to plasma proteins (PPB values greater than 90%), resulting in a low therapeutic
index. However, the results obtained for VD and BBB were appropriate for all tested
compounds. In terms of excretion, isoorientin and ursolic acid showed low clearance (CL),
while α-amyrin and chrysin exhibited good clearance. The half-life (T1/2) was long for
α-amyrin and ursolic acid but short for chrysin and isoorientin.
Regarding toxicity, none of the compounds were found to be probable inhibitors of
the human ether-a-go-go-related gene (hERG) channels or carcinogenic. However, only
ursolic acid presented an intermediate probability of being hepatotoxic to humans (H-HT).

3. Discussion
Malaria is an infectious disease caused by the protozoa parasite Plasmodium spp. and
transmitted by mosquitoes of the Anopheles spp. genus [23]. Despite numerous efforts,
the World Health Organization (WHO) acknowledges that insufficient progress has been
made towards its eradication [24]. Therefore, it is crucial to continue the search for new
compounds that exhibit enhanced activity against the parasite while minimizing side
effects [25]. Plants remain an important source for discovering biologically active com-
pounds of interest [26–28]. Hence, in this study, we focused on identifying and evaluating
the antimalarial activity of specific compounds present in C. obtusifolia compounds such
as α-amyrin, ursolic acid, chrysin, and isoorientin; the degree of purity of a compound,
Molecules 2023, 28, 6912 8 of 14

identifying and comparing analytes, and monitoring a reaction. It is easy to understand

and execute, allowing separations in a short time and them to be versatile and low cost.
In addition, only small amounts of reagents, standards, and samples are used for the
identification of different compounds even in the same run [29,30]. These compounds
have been previously reported in research involving species belonging to the Cecropeacea
family, as demonstrated by studies such as Costa et al., 2011 [13], Rivera-Mondragon et al.,
2017 [14], and others.
To evaluate the antimalarial activity, we utilized computer-aided drug design (CADD)
tools. CADD encompasses various techniques such as de novo drug design, receptor-
based ab initio pharmacophore modeling, dynamic trajectory water pharmacophores, free
energy perturbation calculations, polypharmacology, and big data analysis [31–33]. One
of these tools is molecular docking, which allows for the assessment of binding affinity
between a target protein and a chemical compound of interest, thereby predicting the
compound’s ability to bind effectively to the protein [34–36]. In our study, we evaluated
the binding affinity between the four compounds identified in C. obtusifolia and three
pharmacologically relevant proteins from Plasmodium spp.: dihydrofolate reductase protein
(2BL9) [17], aspartic protease (4ZL4) known as Plasmepsin V, both from P. vivax [18], and
lactate dehydrogenase (1CET) from P. falciparum [21].
Regarding the targets of interest, dihydrofolate reductase (2BL9) is a target of interest
for the treatment of various infectious diseases, including leishmaniasis, tuberculosis,
bacterial infections, and malaria, among others [37]. It plays a crucial role alongside
thymidylate synthase in the recycling of folates, which is important for the synthesis
of purines and methionine [38]. Upon infecting erythrocytes, Plasmodium spp. releases
numerous proteins into the cytosol that are essential for its survival in this environment.
One such vital enzyme in these pathways is the aspartic protease plasmepsin V (4ZL4) [39].
Additionally, this enzyme plays a significant role during gametocytogenesis. On another
note, lactate dehydrogenase (1CET) functions primarily in the conversion of pyruvate to
lactate, a crucial process for energy production in most living organisms. In the asexual
stages of P. falciparum, lactate dehydrogenase produces lactate as a byproduct associated
with growth regulation [40]. This enzyme is considered an important drug target in malaria
treatment and inhibiting its activity leads to the parasite’s demise.
Having compounds that can inhibit this type of enzyme is a key aspect of the develop-
ment of new antimalarial drugs [41–43]. Therefore, in this study, we evaluated the ability
of C. obtusifolia compounds to bind to and inhibit these enzymes and determined their po-
tential as antimalarial agents. Our compounds exhibited greater binding affinity compared
to the control compounds obtained from the provided .pdb file, except for the 4ZL4-ursolic
acid complex. Among the compounds tested, chrysin demonstrated the highest affinity for
both 2BL9 (−8.7 kcal/mol) and 4ZL4 (−9.6 kcal/mol); similar results to those obtained by
Sivaramakrishnan et al. [44] against Plasmepsin V and Chaniad et al. [45] against P. falci-
parum lactate dehydrogenase. Chrysin is a natural polyphenol with documented biological
activities, including anti-cancer and hepatoprotective properties [46]. Likewise, it has been
verified that in vitro it inhibits the enzymes Falcipain-2 and Plasmepsin II [47] to an extent
between 24 and 35%; it also showed activity in vitro assays against strains of P. falciparum
sensitive and resistant to chloroquine [48]. On the other hand, isoorientin is a C-glucosyl
flavone with verified biological activities, such as reducing hyperglycemia, hyperlipidemia,
insulin resistance [49,50], anti-inflammatory effects [51], and activity against certain types
of cancer [52,53]. Its antimalarial potential has also been evaluated [54], although its spe-
cific mechanism of action remains undetermined. Based on these findings, we propose
that chrysin and isoorientin could be effective antimalarial drugs as they bind effectively
to both P. vivax and P. falciparum receptors, exhibiting lower binding energies than the
control compounds.
To assess the stability of the evaluated complexes from molecular docking, a molecular
dynamics (MD) analysis was conducted on the best docking results. MD simulations
investigate the dynamic behavior of proteins and their interactions with small molecules,
Molecules 2023, 28, 6912 9 of 14

such as drugs [55]. To confirm the stability of the analyzed complexes, several parameters
including RMSD, RMSF, RG, and SASA were calculated.
RMSD, or root mean square displacement, measures the average deviation of backbone
atoms in the protein–ligand complex from a reference structure (Rref) and is plotted over
the simulation time. RMSF, or root mean square fluctuations, quantify the positional
deviations of individual particles (atoms) in relation to their reference positions. The radius
of gyration (RG) provides information about the overall size and structural changes of
the protein during MD simulations [44]. The solvent accessible surface area (SASA) is a
significant criterion that indicates the extent of receptor exposure to surrounding solvent
molecules during the simulation [56].
By analyzing these parameters, we confirmed that chrysin and isoorientin do not
significantly impact the stability of the studied targets. They remain attached to the
proteins without causing structural disturbances, suggesting their potential to function
effectively when administered and bound to specific protozoan proteins.
In order for a substance to be used as a medicine, it must undergo evaluations to
determine its ability to efficiently navigate through the body, thereby producing the ex-
pected effects. Additionally, the substance’s potential toxicity needs to be evaluated. These
assessments fall under the domain of ADMET, which encompasses pharmacokinetics [57].
Conducting such evaluations can be expensive and time-consuming; however, there are
currently cheminformatics tools available that enable the prediction of chemical substance
behavior within an organism [58]. In our study, we utilized the ADMETlab 2.0 platform to
predict the pharmacokinetic parameters of the four compounds identified in C. obtusifolia.
ADMETlab 2.0 is a web server designed for predicting the pharmacokinetics and toxicity
properties of chemicals, including various endpoints such as physicochemical properties,
medicinal chemistry properties, ADME properties, toxicity endpoints, and toxicophore
rules [59].
Among the four compounds, isoorientin is the only one that may not be suitable
for oral administration as it exhibits low intestinal absorption and poor permeability
through Caco-2 cells. However, the possibility of administering it via non-oral routes is
not ruled out. Distribution analysis indicates favorable volume distribution (VD) and
high affinity to plasma protein binding (PPB) for all compounds, as well as the ability to
penetrate the blood–brain barrier (BBB). This last aspect is particularly relevant for cases
of cerebral malaria, as certain drugs may struggle to reach the brain due to this biological
barrier [60,61].
Regarding excretion, α-amyrin demonstrates the most favorable values for both clear-
ance and half-life (T1/2). However, the excretion values of the other compounds do not
impose limitations on their potential administration; rather, dosage adjustments may be
necessary based on their respective rates of excretion [62,63].
Toxicity is a critical consideration when assessing compounds for potential drug use
in vivo. Therefore, it is crucial to determine any potential toxicity before initiating further
analyses [64]. One of the primary concerns in this type of assessment is the inhibition of
hERG channels which can lead to irregular heart rhythm and even cardiovascular arrest [65].
Fortunately, based on our results, none of the compounds exhibit hERG channel inhibition
nor do they display carcinogenic potential. However, it should be noted that ursolic acid
shows an intermediate probability of being hepatotoxic to humans.

4. Materials and Methods

4.1. Proteins Preparation
Crystal structures of key receptors for P. vivax (2BL9, 4ZL4) and P. falciparum (1CET)
were obtained from the Protein Data Bank (http://www.rcsb.org/, accessed on 25 Novem-
ber 2022) with resolutions of 1.90 Å, 2.37 Å, and 2.05 Å, respectively. The structures were
processed using the Dock Prep Module of UCSF Chimera 1.14 [66]. This involved removing
water molecules, additional chains, and ligands, as well as adding hydrogen atoms and
Molecules 2023, 28, 6912 10 of 14

assigning partial charges. Any missing protein fragments were reconstructed with the
assistance of I-TASSER [67].

4.2. Compound Retrieval and Preparation

The ligands (ursolic acid, α-amyrin, chrysin, and isoorientin) and control compounds
(chloroquine, pyrimethamine, and WEHI-842) were obtained in a Mole2 file format from
PubChem (https://pubchem.ncbi.nlm.nih.gov/, accessed on 25 November 2022). Avo-
gadro 1.2.0 [68] was used for geometry optimization of the ligands and to convert the file
format to .pdb. The prepared ligands were then used with the Chimera docking tool.

4.3. Molecular Docking Analysis

All structures were aligned and a large grid box accommodating all experimental
ligands was used. A grid box of 25 × 25 × 25 Å was employed for all structures. The
coordinates of the grid box for each target were as follows: 2BL9 (64.00, 70.00, and 60.38),
4ZL4 (70.99, 71.13, and 70.93), and 1CET (60.903, 75.211, and 50.674), based on the previous
position of the natural ligand and providing additional space. The Lamarckian genetic
algorithm (LGA) was performed with the rigid receptor molecule to search for the best
conformers. Ten independent docking experiments were carried out for each structure using
the Autodock Vina standalone tool [69,70]. Among the ten replicates, the most frequently
recurring energy and pose were selected as the final result, which were visualized using
LigPlot+ [71] and PyMOL [72]. The docking protocol for each protein was validated by
superimposing the re-docked poses of co-crystallized ligands with the crystal conformations
extracted from the PDB and the RMSD was calculated in PyMOL. The RMSD value should
fall within a reliable range of 2 Å [M].

4.4. Molecular Dynamics Simulation

Molecular dynamics simulations were conducted to assess the binding stability, con-
formation, and interaction modes between the compounds (ursolic acid, α-amyrin, chrysin,
and isoorientin) and receptors (1CET, 2BL9, and 4ZL4). The selected ligand–receptor
complex files were subjected to molecular dynamics studies using GROMACS 2019.2
software [73]. The topologies of the selected ligands were obtained from the PRODRG
server [74].
For the MD simulation, the complex structure was first minimized in a vacuum using
the steepest descent algorithm for 5000 steps. The solvated complex was then placed in
a cubic periodic box with dimensions of 0.5 nm, using a simple point charge (SPC) water
model and maintained at a temperature of 310 K. The system was further equilibrated with
an appropriate salt concentration of 0.15 M by adding Na+ and Cl− ions. Each complex
underwent a 50 ns simulation in the NPT ensemble (isothermal–isobaric, constant number
of particles, pressure, and temperature).
Trajectory analysis, including root mean square deviation (RMSD), root mean square
fluctuation (RMSF), radius of gyration (RG), and solvent accessible surface area (SASA), was
performed using the GROMACS simulation package through the online server “WebGRO
for Macromolecular Simulations” (https://simlab.uams.edu/, accessed on 5 January 2023).
Graphs were generated using the Xmgrace tool of Grace Software [75] and Microsoft Excel.

4.5. Pharmacokinetic Properties

In silico pharmacokinetic and toxicity analyses of the compounds were conducted
using ADMETlab 2.0 [57], a free online web server for analyzing ADMET properties. The
isomeric SMILE codes of the compounds were entered into the server.

5. Conclusions
This study demonstrates the potential inhibitory activity of the four compounds
identified in C. obtusifolia against important proteins of the Plasmodium genus (2BL9,
4ZL4, and 1CET). Specifically, α-amyrin and isoorientin exhibited the most promising
Molecules 2023, 28, 6912 11 of 14

activities and their pharmacokinetic behavior was predicted to be satisfactory, indicating

their potential for administration without issues. Therefore, these two compounds possess
the necessary characteristics, along with in vitro and in vivo studies, to be considered as
potential antimalarial agents. Moreover, these results provide evidence supporting the
traditional use of C. obtusifolia as a treatment for malaria.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules28196912/s1, Table S1: All pharmacokinetics results
from ADMETlab 2.0.
Author Contributions: Conceptualization, C.A.L.-T.; Investigation, Z.E.O.-V.; Methodology, Y.M.-H.
and C.A.L.-T.; Software, C.A.L.-T.; Supervision, Z.E.O.-V.; Writing—original draft, Z.E.O.-V., Y.M.-H.
and C.A.L.-T.; Writing—review and editing, Z.E.O.-V., Y.M.-H. and C.A.L.-T. All authors have read
and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: For data supporting the reported results, please contact the corres-
ponding author.
Acknowledgments: We thank CONACyT for the scholarship No. 516001 awarded to YMH.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not applicable.

References
1. World Health Organization. Informe Mundial Sobre el Paludismo 2021; World Health Organization: Geneva, Switzerland, 2021.
2. Organización Mundial de la Salud. Malaria. 2023. Available online: https://www.who.int/es/news-room/fact-sheets/detail/
malaria (accessed on 29 March 2023).
3. Pereira, Á.; Ríos, M.P. Epidemiología y Tratamiento del Paludismo. OFFARM 2002, 21, 110–116.
4. Rodríguez, M.H.; Betanzos-Reyes, Á.F. Plan de mejoramiento del control de la malaria hacia su eliminación en Mesoamérica.
Salud Pública México 2011, 53, 333–348.
5. Organización Mundial de la Salud. Informe Mundial Sobre la Malaria 2021 OMS. 2021. Available online: https://cdn.who.int/me
dia/docs/default-source/malaria/world-malaria-reports/world-malaria-report-2021-regional-briefing-kit-spa.pdf?sfvrsn=338
167b6_25&download=true (accessed on 29 March 2023).
6. Boletin Epiodemiológico de la Secretaria de Salud. Situación Epidemiológica de Malaria en México, Hasta la Semana 27 del 2020.
Available online: http://www.anhp.org.mx/archivoseventos/03-Panorama-Epidemiol%C3%B3gico-Paludismo_16-07-2020.pdf
(accessed on 21 August 2021).
7. Alonso-Castro, A.J.; Miranda-Torres, A.C.; González-Chávez, M.M.; Salazar-Olivo, L.A. Cecropia obtusifolia Bertol and its active
compound, chlorogenic acid, stimulate 2-NBDglucose uptake in both insulin-sensitive and insulin-resistant 3T3 adipocytes.
J. Ethnopharmacol. 2008, 120, 458–464. [CrossRef] [PubMed]
8. Vidal Durango, J.; Marrugo Negrete, J.; Jaramillo Colorado, B.; Pérez Castro, L. Remediation of soils contaminated with mercury
using guarumo (Cecropia peltata). Eng. Dev. Univ. North 2010, 27, 113–129.
9. Medrano-Sánchez, E.J.; Hernández-Bolio, G.I.; Lobato-García, C.E.; González-Cortazar, M.; Antunez-Mojica, M.; Gallegos-García,
A.J.; Barredo-Hernández, C.O.; López-Rodríguez, R.; Aguilar-Sánchez, N.C.; Gómez-Rivera, A. Intra- and Interspecies Differences
of Two Cecropia Species from Tabasco, Mexico, Determined through the Metabolic Analysis and 1 H-NMR-Based Fingerprinting of
Hydroalcoholic Extracts. Plants 2023, 12, 2440. [CrossRef]
10. Trejo, G.M. Phytochemical Study of Guarumbo (Cecropia obtusifolia) as a Hypoglycemic Agent. Bachelor’s Thesis, National
Polytechnic Institute (IPN), National School of Biological Sciences (ENCB), La Ciudad de México, Mexico, 1983; pp. 1–55.
11. Sanchez, M.B.; Miranda-Pérez, E.; Verjan, J.C.G.; de Los Angeles Fortis Barrera, M.; Pérez-Ramos, J.; Alarcón-Aguilar, F.J. Potential
of the chlorogenic acid as multitarget agent: Insulin-secretagogue and PPAR α/γ dual agonist. Biomed. Pharmacother. 2017,
94, 169–175. [CrossRef]
12. Cadena-Zamudio, J.D.; Nicasio-Torres, P.; Monribot-Villanueva, J.L.; Guerrero-Analco, J.A.; Ibarra-Laclette, E. Integrated Analysis
of the Transcriptome and Metabolome of Cecropia obtusifolia: A Plant with High Chlorogenic Acid Content Traditionally Used to
Treat Diabetes Mellitus. Int. J. Mol. Sci. 2020, 21, 7572. [CrossRef]
13. Costa, G.M.; Schenkel, E.P.; Reginatto, F.H. Chemical and pharmacological aspects of the genus Cecropia. Nat. Prod. Common.
2011, 6, 913–920. [CrossRef]
Molecules 2023, 28, 6912 12 of 14

14. Rivera-Mondragón, A.; Tuenter, E.; Bijttebier, S.; Cos, P.; Apers, S.; Caballero-George, C.; Foubert, K.; Pieters, L. Two new
antiplasmodial flavonolignans from the leaves of Cecropia obtusifolia. Phytochem. Lett. 2019, 31, 118–120. [CrossRef]
15. Bilsland, E.; Van Vliet, L.; Williams, K.; Feltham, J.; Carrasco, M.P.; Fotoran, W.L.; Cubillos, E.F.G.; Wunderlich, G.; Grøtli, M.;
Hollfelder, F.; et al. Plasmodium dihydrofolate reductase is a second enzyme target for the antimalarial action of triclosan. Sci. Rep.
2018, 8, 1038. [CrossRef]
16. Sharma, D.; Lather, M.; Mallick, P.K.; Adak, T.; Dang, A.S.; Valecha, N.; Sing, O.P. Polymorphism in drug resistance genes
dihydrofolate reductase and dihydropteroate synthase in Plasmodium falciparum in some states of India. Parasit. Vectors 2015,
8, 471. [CrossRef] [PubMed]
17. Kongsaeree, P.; Khongsuk, P.; Leartsakulpanich, U.; Chitnumsub, P.; Tarnchompoo, B.; Walkinshaw, M.D.; Yuthavong, Y. Crystal
Structure of Dihydrofolate Reductase from Plasmodium Vivax: Pyrimethamine Displacement Linked with Mutation-Induced
Resistance. Proc. Natl. Acad. Sci. USA 2005, 102, 13046. [CrossRef] [PubMed]
18. Hodder, A.N.; Sleebs, B.E.; Czabotar, P.E.; Gazdik, M.; Xu, Y.; O’Neill, M.T.; Lopaticki, S.; Nebl, T.; Triglia, T.; Smith, B.J.; et al.
Structural basis for plasmepsin V inhibition that blocks export of malaria proteins to human erythrocytes. Nat. Struct. Mol. Biol.
2015, 22, 590–596. [CrossRef] [PubMed]
19. Singh, R.; Bhardwaj, V.; Purohit, R. Identification of a novel binding mechanism of Quinoline based molecules with lactate
dehydrogenase of Plasmodium falciparum. J. Biomol. Struct. 2019, 39, 348–356. [CrossRef]
20. Liu, P. Plasmepsin: Function, characterization and targeted antimalarial drug development. In Natural Remedies in the Fight
Against Parasites; Books on Demand: Norderstedt, Germany, 2017; pp. 183–218.
21. Read, J.A.; Wilkinson, K.W.; Tranter, R.; Sessions, R.B.; Brady, R.L. Chloroquine binds in the cofactor binding site of Plasmodium
falciparum lactate dehydrogenas. J. Biol. Chem. 1999, 274, 10213–10218. [CrossRef]
22. Plucinski, M.M.; McElroy, P.D.; Dimbu, P.R.; Fortes, F.; Nace, D.; Halsey, E.S.; Rogier, E. Clearance dynamics of lactate dehy-
drogenase and aldolase following antimalarial treatment for Plasmodium falciparum infection. Parasites Vectors 2019, 12, 293.
[CrossRef]
23. Arora, G.; Chuang, Y.; Sinnis, P.; Dimopoulos, G.; Fikrig, E. Malaria: Influence of Anopheles mosquito saliva on Plasmodium infection.
Trends Immunol. 2023, 44, 256–265. [CrossRef]
24. WHO. Report of the WHO Strategic Advisory Group on Malaria Eradication; A Report of the Strategic Advisory Group on Malaria
Eradication; World Health Organization: Geneva, Switzerland, 2020.
25. Van der Plujim, R.W.; Amaratunga, C.; Dhorda, M.; Dondorp, A.M. Triple Artemisinin-Based Combination Therapies for
Malaria—A New Paradigm? Trends Parasitol. 2021, 37, 15–24. [CrossRef]
26. Zhao, Y.; Wu, Y.; Wang, M. Bioactive Substances of Plant Origin. In Handbook of Food Chemistry; Cheung, P.C.K., Mehta, B.M., Eds.;
Springer Nature: Berlin/Heidelberg, Germany, 2020; pp. 967–1008.
27. Qasim, M.; Abideen, Z.; Adnan, M.; Gulzar, S.; Gul, B.; Rasheed, M.; Khan, M. Antioxidant properties, phenolic composition,
bioactive compounds and nutritive value of medicinal halophytes commonly used as herbal teas. S. Afr. J. Bot. 2017, 110, 240–250.
[CrossRef]
28. Chandran, H.; Meena, M.; Barupal, T.; Sharma, K. Plant tissue culture as a perpetual source for production of industrially
important bioactive compounds. Biotechnol. Rep. 2020, 26, e00450. [CrossRef]
29. Jug, G.; Anderluh, M.; Tomašič, T. Comparative evaluation of several docking tools for docking small molecule ligands to
DC-SIGN. J. Mol. Model. 2015, 21, 164. [CrossRef] [PubMed]
30. Vallejo-Rosero, Y.J.; Barrios-Correa, L.; Anaya-Gil, J. La cromatografía en capafina: Una alternativa vigente en la industria
farmacéutica. Rev. Química PUCP 2021, 35, 19–25.
31. Yu, W.; Mackerell, A. Computer-aided drug design methods. Methods Mol. Biol. 2017, 1520, 85–106. [PubMed]
32. Prieto-Martínez, F.; Medina-Franco, J. Computer-aided drug design: When informatics, chemistry and art meets. Tip. Rev. Espec.
Cienc. 2018, 21, 124–134.
33. Anwar, T.; Kumar, P.; Khan, A. Modern Tools and Techniques in Computer-Aided Drug Design. Curr. Comput. Aided Drug Des.
2021, 1–30. [CrossRef]
34. Pinzi, L.; Rastelli, G. Molecular Docking: Shifting Paradigms in Drug Discovery. Int. J. Mol. Sci. 2019, 20, 4331. [CrossRef]
35. Saikia, S.; Bordoloi, M. Molecular Docking: Challenges, Advances and its Use in Drug Discovery Perspective. Curr. Drug Targets
2019, 20, 501–521. [CrossRef]
36. Stanzione, F.; Giangreco, I.; Cole, J. Use of molecular docking computational tools in drug discovery. Prog. Med. Chem. 2021,
60, 273–343.
37. Chawla, P.; Teli, G.; Gill, R.K.; Narang, R.K. An Insight into Synthetic Strategies and Recent Developments of Dihydrofolate
Reductase Inhibitors. ChemistrySelect 2021, 6, 12101–12145. [CrossRef]
38. Yuthavong, Y.; Tarnchompoo, B.; Vilaivan, T.; Chitnumsub, P.; Kamchonwongpaisan, S.; Charman, S.A.; Matthews, D. Malarial
dihydrofolate reductase as a paradigm for drug development against a resistance-compromised target. Proc. Natl. Acad. Sci. USA
2012, 109, 16823–16828. [CrossRef]
39. Polino, A.J.; Nasamu, A.S.; Niles, J.C.; Goldberg, D.E. Assessment of biological role and insight into druggability of the Plasmodium
falciparum protease plasmepsin V. ACS Infect. Dis. 2020, 6, 738–746. [CrossRef] [PubMed]
Molecules 2023, 28, 6912 13 of 14

40. Correa, R.; Coronado, L.; Caballero, Z.; Faral-Tello, P.; Robello, C.; Spadafora, C. Extracellular vesicles carrying lactate dehy-
drogenase induce suicide in increased population density of Plasmodium falciparum in vitro. Sci. Rep. 2019, 9, 5042. [CrossRef]
[PubMed]
41. Hawser, S.; Lociuro, S.; Islam, K. Dihydrofolate reductase inhibitors as antibacterial agents. Biochem. Pharmacol. 2006, 71, 941–948.
[CrossRef] [PubMed]
42. Polino, A.J.; Miller, J.J.; Bhakat, S.; Mukherjee, S.; Bobba, S.; Bowman, G.R.; Goldberg, D.E. The nepenthesin insert in the
Plasmodium falciparum aspartic protease plasmepsin V is necessary for enzyme function. J. Biol. Chem. 2022, 298, 102355.
[CrossRef]
43. Shadrack, D.M.; Nyandoro, S.S.; Munissi, J.J.; Mubofu, E.B. In silico evaluation of anti-malarial agents from Hoslundia opposita
as inhibitors of Plasmodium falciparum lactate dehydrogenase (PfLDH) enzyme. Comput. Mol. Biosci. 2016, 6, 23–32. [CrossRef]
44. Sivaramakrishnan, M.; Kandaswamy, K.; Natesan, S.; Devarajan, R.; Ramakrishnan, S.; Kothandan, R. Molecular docking and
dynamics studies on plasmepsin V of malarial parasite Plasmodium vivax. Inform. Med. Unlocked 2020, 19, 100331. [CrossRef]
45. Chaniad, P.; Mungthin, M.; Payaka, A.; Viriyavejakul, P.; Punsawad, C. Antimalarial properties and molecular docking analysis
of compounds from Dioscorea bulbifera L. as new antimalarial agent candidates. BMC Complement. Med. Ther. 2021, 21, 144.
[CrossRef]
46. Stompor-goracy,˛ M.; Bajek-bil, A.; Machaczka, M. Chrysin: Perspectives on Contemporary Status and Future Possibilities as
Pro-Health Agent. Nutrients 2021, 13, 2038. [CrossRef]
47. Jin, H.; Xu, Z.; Cui, K.; Zhang, T.; Lu, W.; Huang, J. Dietary flavonoids fisetin and myricetin: Dual inhibitors of Plasmodium
falciparum falcipain-2 and plasmepsin II. Fitoterapia 2014, 94, 55–61. [CrossRef]
48. Lehane, A.M.; Saliba, K.J. Common dietary flavonoids inhibit the growth of the intraerythrocytic malaria parasite. BMC Res.
Notes 2008, 18, 26. [CrossRef]
49. Ziqubu, K.; Dludla, P.; Joubert, E.; Muller, C.; Louw, J.; Tiano, L.; Nkambule, B.; Kappo, A.; Mazibuko-Mbeje, S. Isoorientin: A
dietary flavone with the potential to ameliorate diverse metabolic complications. Pharmacol. Res. 2020, 158, 104867. [CrossRef]
[PubMed]
50. Ziqubu, K.; Muller, C.; Dludla, P.; Mthembu, S.; Obonye, N.; Louw, J.; Kappo, A.; Silvestri, S.; Orlando, P.; Tiano, L.; et al. Impact
of Isoorientin on Metabolic Activity and Lipid Accumulation in Differentiated Adipocytes. Molecules 2020, 25, 1773. [CrossRef]
[PubMed]
51. Anilkumar, K.; Reddy, G.; Azad, R.; Yarla, N.; Dharmapuri, G.; Srivastava, A.; Kamal, M.; Pallu, R. Evaluation of Anti-
Inflammatory Properties of Isoorientin Isolated from Tubers of Pueraria tuberosa. Oxid. Med. Cell Longev. 2017, 2017, 5498054.
[CrossRef] [PubMed]
52. Lin, X.; Wei, J.; Chen, Y.; He, P.; Lin, J.; Tan, S.; Nie, J.; Lu, S.; He, M.; Lu, Z.; et al. Isoorientin from Gypsophila elegans induces
apoptosis in liver cancer cells via mitochondrial-mediated pathway. J. Ethnopharmacol. 2016, 187, 187–194. [CrossRef] [PubMed]
53. Zhang, T.; Zhang, Y.; Wang, S.; Wang, J.; Luo, Y.; Jin, C. Research progress on anti-tumor pharmacological effects and mechanism
of isoorientin. Anim. Feed. Sci. 2019, 40, 86–88.
54. Atay, I.; Kirmizibekmez, H.; Kaiser, M.; Akaydin, G.; Yesilada, E.; Tasdemir, D. Evaluation of in vitro antiprotozoal activity of
Ajuga laxmannii and its secondary metabolites. Pharm. Biol. 2016, 54, 1808–1814. [CrossRef]
55. Salo-Ahen, O.; Alanko, I.; Bhadane, R.; Alexandre, A.; Honorato, R.; Hossain, S.; Juffer, A.; Kabedev, A.; Lahtela-Kakkonen, M.;
Larsen, A.; et al. Molecular Dynamics Simulations in Drug Discovery and Pharmaceutical Development. Processes 2021, 9, 71.
[CrossRef]
56. Kalimuthu, A.; Panneerselvam, T.; Pavadai, P.; Pandian, S.; Sundar, K.; Murugesan, S.; Ammunje, D.; Kumar, S.; Arunachalam, S.;
Kunjiappan, S. Pharmacoinformatics-based investigation of bioactive compounds of Rasam (South Indian recipe) against human
cancer. Sci. Rep. 2021, 11, 21488. [CrossRef]
57. Van den Anker, J.; Reed, M.; Allegaert, K.; Kearns, G. Developmental Changes in Pharmacokinetics and Pharmacodynamics.
J. Clin. Pharmacol. 2018, 58, S10–S25. [CrossRef]
58. Parikh, P.; Savjani, J.; Gajjar, A.; Chhabria, M. Bioinformatics and Cheminformatics Tools in Early Drug Discovery. In Bioinformatics
Tools for Pharmaceutical Drug Product Development; Chavda, V., Anand, K., Apostolopoulos, V., Eds.; John Wiley & Sons: Hoboken,
NJ, USA, 2023.
59. Xiong, G.; Wu, Z.; Yi, J.; Fu, L.; Yang, Z.; Hsieh, C.; Yin, M.; Zeng, X.; Wu, C.; Lu, A.; et al. ADMETlab 2.0: An integrated online
platform for accurate and comprehensive predictions of ADMET properties. Nucleic Acids Res. 2021, 49, W5–W14. [CrossRef]
60. Alqahtani, M.; Kazi, M.; Alsenaidy, M.; Ahmad, M. Advances in Oral Drug Delivery. Front. Pharmacol. 2021, 12, 618411. [CrossRef]
[PubMed]
61. Rénia, L.; Howland, S.; Claser, C.; Gruner, A.; Suwanarusk, R.; Teo, T.; Russell, B.; Lisa, N. Cerebral malaria: Mysteries at the
blood-brain barrier. Virulence 2012, 3, 193–201. [CrossRef] [PubMed]
62. Lucas, A.; Sproston, J.; Barton, P.; Riley, R. Estimating human ADME properties, pharmacokinetic parameters and likely clinical
dose in drug discovery. Expert. Opin. Drug Discov. 2019, 14, 1313–1327. [CrossRef] [PubMed]
63. Li, Y.; Meng, Q.; Yang, M.; Liu, D.; Hou, X.; Tang, L.; Wang, X.; Lyu, Y.; Chen, X.; Liu, K.; et al. Current trends in drug metabolism
and pharmacokinetics. Acta Pharm. Sin. B 2019, 9, 1113–1144. [CrossRef]
64. Basile, A.; Yahi, A.; Tatonetti, N. Artificial Intelligence for Drug Toxicity and Safety. Trends Pharmacol. 2019, 40, 624–635. [CrossRef]
Molecules 2023, 28, 6912 14 of 14

65. Garrido, A.; Lepailleur, A.; Mignani, S.; Dallemagne, P.; Rochais, C. hERG toxicity assessment: Useful guidelines for drug design.
Eur. J. Med. Chem. 2020, 195, 112290. [CrossRef]
66. Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF Chimera--a visualization
system for exploratory research and analysis. Comput. Chem. 2004, 25, 1605–1612. [CrossRef]
67. Zhou, X.; Zheng, W.; Li, Y.; Pearce, R.; Zhang, C.; Bell, E.; Zhang, G.; Zhang, Y. I-TASSER-MTD: A deep-learning-based platform
for multi-domain protein structure and function prediction. Nat. Protoc. 2022, 17, 2326–2353. [CrossRef]
68. Hanwell, M.; Curtis, D.; Lonie, D.; Vandermeerschd, T.; Zurek, E.; Hutchison, G. Avogadro: An advanced semantic chemical
editor, visualization, and analysis platform. J. Cheminformatics 2012, 4, 17. [CrossRef]
69. Eberhardt, J.; Santos-Martins, D.; Tillack, A.; Forli, S. AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and
Python Bindings. J. Chem. Inf. Model. 2021, 61, 3891–3898. [CrossRef]
70. Trott, O.; Olson, A. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient
optimization, and multithreading. J. Comput. Chem. 2010, 31, 455–461. [CrossRef] [PubMed]
71. Laskowski, R.A.; Swindells, M.B. LigPlot+: Multiple ligand-protein interaction diagrams for drug discovery. J. Chem. Inf. Model.
2011, 51, 2778–2786. [CrossRef] [PubMed]
72. DeLano, W.L. Pymol: An open-source molecular graphics tool, CCP4 Newsletter. Protein Crystallogr. 2002, 40, 82–92.
73. Páll, S.; Zhmurov, A.; Bauer, P.; Abraham, M.; Lundborg, M.; Gray, A.; Hess, B.; Lindahl, E. Heterogeneous parallelization and
acceleration of molecular dynamics simulations in GROMACS. J. Chem. Phys. 2020, 153, 134110. [CrossRef]
74. Van Aalten, D.M. PRODRG, a program for generating molecular topologies and unique molecular descriptors from coordinates
of small molecules. J. Comput. Aided Mol. Des. 1996, 10, 255–262. [CrossRef]
75. Turner, P.J. XMGRACE, Version 5.1.19; Center for Coastal and Land-Margin Research, Oregon Graduate Institute of Science and
Technology: Beaverton, OR, USA, 2005.

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