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Antimalarial potential, LC-MS secondary metabolite profiling and

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Antimalarial potential, LC–MS secondary

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Kolade O. Faloye, Manish K. Tripathi, Stephen A. Adesida, Samuel A.

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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2023.2205949

Antimalarial potential, LC–MS secondary metabolite profiling and computational

studies of Zingiber officinale
Kolade O. Faloyea, Manish K. Tripathib, Stephen A. Adesidac, Samuel A. Oguntimehind, Yemisi M. Oyetunded,
Adetola H. Adewolee , Ifeoluwa I. Ogunlowoc, Esther A. Idowuc, Uduak I. Olayemic and Olamide D. Dosumuf
a
Department of Chemistry, Faculty of Science, Obafemi Awolowo University, Ile-Ife, Nigeria; bPharmaceutical Chemistry Research Laboratory,
Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (Banaras Hindu University), Varanasi, India;
c
Department of Pharmacognosy, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria; dDepartment of Pharmacognosy, Faculty
of Pharmacy, University of Ibadan, Ibadan, Nigeria; eDepartment of Chemistry, University of Pretoria, Pretoria, South Africa; fDepartment of
Botany, Faculty of Science, Obafemi Awolowo University, Ile-Ife, Nigeria
Communicated by Ramaswamy H. Sarma

ABSTRACT ARTICLE HISTORY

Malaria is among the top-ranked parasitic diseases that pose a threat to the existence of the human Received 25 October 2022
race. This study evaluated the antimalarial effect of the rhizome of Zingiber officinale in infected mice, Accepted 17 April 2023
performed secondary metabolite profiling and detailed computational antimalarial evaluation through
KEYWORDS
molecular docking, molecular dynamics (MD) simulation and density functional theory methods. The
Density functional theory;
antimalarial potential of Z. officinale was performed using the in vivo chemosuppressive model; sec- Liquid chromatography–
ondary metabolite profiling was carried out using liquid chromatography–mass spectrometry (LC–MS). mass spectrometry; malaria;
Molecular docking was performed with Autodock Vina while the MD simulation was performed with molecular docking;
Schrodinger desmond suite for 100 ns and DFT calculations with B3LYP (6-31G) basis set. The extract molecular dynamics
showed 64% parasitaemia suppression, with a dose-dependent increase in activity up to 200 mg/kg. simulation; Zingiber
The chemical profiling of the extract tentatively identified eight phytochemicals. The molecular dock- officinale
ing studies with plasmepsin II and Plasmodium falciparum dihydrofolate reductase-thymidylate syn-
thase (PfDHFR-TS) identified gingerenone A as the hit molecule, and MMGBSA values corroborate the
binding energies obtained. The electronic parameters of gingerenone A revealed its significant anti-
malarial potential. The antimalarial activity elicited by the extract of Z. officinale and the bioactive
chemical constituent supports its usage in ethnomedicine.

1. Introduction plasmepsins (Bhaumik, Gustchina, and Wlodawer 2012; Liu

Malaria is a parasitic disease that manifests in humans and is
caused by Plasmodium parasites transmitted to humans
through the bite of an infected female Anopheles mosquito
(Cator et al. 2012; Yadav, Tripathi, and Yadav 2021). It is a
severe and often deadly disease, especially when the parasite
involved is Plasmodium falciparum (Jensen, Adams, and Hviid
2020). Despite various interventions in treatment and preven-
tion, about 241 million new cases and 627,000 deaths were
attributed to malaria in 2020 (WHO, 2022). This same year,
African countries accounted for 96% of the reported deaths
due to malaria, 31.9% of which occurred in Nigeria (WHO,
2021). Clinical symptoms of malaria, such as high fever, chills
and rigours, flu-like illness, head, muscle and joint aches,
among others, occur when the parasite is in the blood stage
of its cycle in the human host (Rudrapal and Chetia, 2017).
The roles of some enzymes in the pathophysiology of
malaria cannot be underestimated. The initial degradation of
hemoglobin in the food vacuole of the malaria parasite to
obtain nutrients is catalyzed by aspartic proteases known as
2017). Four of the ten plasmepsins including plasmepsin II,
encoded by the genome of P. falciparum are associated with
its food vacuole, which is directly related to the role of the
parasite in the pathophysiology of malaria (Chugh et al.
2013). Another important enzyme implicated in malaria is P.
falciparum dihydrofolate reductase-thymidylate synthase
(PfDHFR-TS) which converts dihydrofolate to tetrahydrofolate
and is involved in the synthesis of purines and thymidylate,
all of which are important in DNA/RNA synthesis and repair,
cell division and protein synthesis (Sharma and Chauhan
2012; Shamshad, Bakri, and Mirza 2022). These actions by
PfDHFR-TS make it a prime target in antifolate antimalarial
therapy (Yuthavong et al. 2005). Therefore, inhibiting the
activities of PfDHFR-TS and Plasmepsin II is an effective strat-
egy in the research and development of novel drug mole-
cules for antimalarial therapy.
Z. officinale Roscoe is a plant in the family Zingiberaceae.
The rhizome of the plant is used as a spice and for its medi-
cinal properties. It is used to treat cough and cold, head-
aches, nausea, flatulence, malaria, and vomiting (Bhowmik

CONTACT Kolade O. Faloye kollintonx1@gmail.com Department of Chemistry, Faculty of Science, Obafemi Awolowo University, Ile-Ife, Nigeria; Uduak I.
Olayemi uduakolayemi@gmail.com Department of Pharmacognosy, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2023.2205949.
ß 2023 Informa UK Limited, trading as Taylor & Francis Group
2 K. O. FALOYE ET AL.
et al. 2010; Vandebroek and Picking 2020). Its antioxidant, anti-
inflammatory, antimicrobial, and anticancer activities have been
reported (Otunola et al. 2010; Yusuf et al. 2018). Kaushik et al.
(2013) reported the in vitro assay of the ethyl acetate solvent
extract of the rhizome of Z. officinale as showing excellent anti-
plasmodial activity (IC50 10 lg/mL); however, Biruksew et al.
(2018) reported the poor antiplasmodial activity elicited by the
methanol root extract of the plant. Nonetheless, neither the
in vivo antiplasmodial activity of the methanol extract of Z. offi-
cinale rhizome nor the computational investigation of the
inhibitory action of its chemical constituents on some key mal-
aria enzymes has been reported.
The drug discovery process takes years of effort and huge
investment to accomplish. Drug targets could be enzymes,
receptors, proteins, or other such molecules that are import-
ant to the disease pathophysiology (Mohs and Greig 2017).
Computational drug discovery is an economical and faster
method used in drug discovery (Faloye et al. 2021).
Molecular docking, pharmacophore modeling and mapping,
de novo design, and molecular simulation are the methods
of computational drug discovery that have found application
in target identification and validation, lead discovery and
optimization, as well as preclinical tests (Ou-Yang et al. 2012;
Sliwoski et al. 2014; Schaduangrat et al. 2020).
With the unavailability of an effective vaccine for the pre-
vention and treatment of malaria and the development of
resistance by P. falciparum to the available antimalarial drugs,
the search for new drug molecules from natural sources is
inevitable (Plowe et al. 1995; Etim et al. 2018). There is a dire
need to evaluate the antimalarial claims of medicinal plants
used in folklore medicine for the treatment of malaria. This
study aims to investigate the in vivo antimalarial activity of
the methanol extract of Z. officinale, perform the chemical
profiling of the methanol extract of Z. officinale, elucidate
the inhibitory action of the chemical constituents on some
enzymes in malaria pathophysiology using molecular docking
and molecular dynamics simulation methods, and estimate
the quantum chemical properties of the hit molecule.
2. Materials and methods
2.1. Plant collection, identification, and extraction
The rhizome of Z. officinale was collected at the Medicinal
Plants Garden, Department of Pharmacognosy, Obafemi
Awolowo University, Ile-Ife, in November 2016. The sample
was identified, and voucher specimens were deposited at
the Forestry Herbarium, Ibadan and the Faculty of Pharmacy
Herbarium, Ife with the codex FHI 110934 and FPI 2356,
respectively. The rhizomes were washed, air-dried, pulverized
and 25 g of the plant material was extracted with methanol
(750 mL) for 72 h. The methanol extract was filtered and con-
centrated in vacuo to afford the crude extract (3 g).
2.2. Animals and parasite
Thirty Swiss mice of either sex (18–22 g) were obtained from
the Animal House, Faculty of Pharmacy, OAU. They were housed
in aluminum cages with wood shavings used as beddings and
allowed free access to water and food under 12 h day/night
cycle. They were acclimatized and cared for according to stand-
ard procedures in the handling of animals (NIH Publication NO
82-85, Revised 1985). The animals were divided into six groups
(I–VI) of five mice each. P. berghei strain NK65 (Chloroquine sen-
sitive) was obtained from the Institute of Advanced Medical
Research and Training (IMRAT), University College Hospital,
Ibadan. The donor mouse was euthanized and blood was with-
drawn through the cardiac puncture into a heparinized bottle.
The blood was diluted such that 0.2 mL of the inoculum con-
tained 107 parasitized red blood cells. Each mouse was inocu-
lated with 0.2 mL of the inoculum via the intraperitoneal route.
2.3. Preparation of solutions of the extract and
standard drug
Appropriate weights of 25, 50, 100, and 200 mg of the
extract of Z. officinale were separately dissolved in 5.0 mL of
normal saline to give the following doses: 50, 100, 200, and
400 mg/kg, respectively. The standard drug (chloroquine) was
prepared by dissolving 8.33 mg of the chloroquine tablet in
5 mL of normal saline to give a dose of 10 mg/kg.
2.4. In vivo antimalarial assays
The chemosuppressive activities were performed by oral admin-
istration of the extract (50 mg/kg, 100 mg/kg, 200 mg/kg and
400 mg/kg) to groups I to IV, and groups V and VI were admin-
istered chloroquine (CQ, 10 mg/kg) and normal saline, respect-
ively, two hours after infection. These were repeated daily for
3 days (Peters 1965). The level of parasitaemia was determined
for each mouse on Day 4 (D4) by taking a thin blood smear
from the tail of each mouse on a slide. The blood smear was
fixed with methanol, stained with Giemsa and counting of para-
sitized blood cells in 5 fields of view on the microscope was car-
ried out (Ryley and Peters 1970). The average parasitaemia in
each group was calculated in order to determine the percent-
age chemosuppressive activity of the extract using the formula:
ðA  BÞ=A  100
where A and B are the mean parasitaemia in the negative
control and the test groups, respectively (Tona et al. 2001).
The extract’s antimalarial chemosuppressive activity was
determined by the percentage reduction of parasitaemia in
treated groups compared with the untreated infected group.
2.5. Survival time
The animals were further observed for 28 days post-treatment
for mortality to determine the survival time of each mouse. The
average survival time for each dose was then calculated in
days ± SEM (Adesida, Odediran, and Elujoba 2021).
2.6. Estimation of the median effective doses ED50 and ED90
A graph of the test doses in mg/kg was automatically plot-
ted against chemosuppression in percentage using Microsoft

Excel 2007. From the graph, the median effective doses
(ED50 and ED90), which are the doses that would give 50%
and 90% chemosuppression, were forecast and recorded as
mg/kg ± SEM.
2.7. Statistical analysis
Values were expressed as mean ± SEM and analyzed statistic-
ally using One-way Analysis of Variance (ANOVA) followed by
Student Newmann Keul’s post-hoc for comparisons to deter-
mine the source of significant difference for all values. Values
of p < 0.05 were of statistical significance.
2.8. LC-MS profiling of methanol extract Z. officinale
rhizome
A linear trap quadrupole-(LTQ) Orbitrap spectrometer
(Thermo Scientific, USA) was used to carry out Liquid chro-
matography–mass spectrometry (LC–MS) analysis. The instru-
ment is equipped with an Agilent 1200 HPLC system (Santa
Clara, CA, USA), and connected to a photodiode array (PDA)
detector. Sample preparation was done by making the rhi-
zome extract into a final concentration of 2 mg/mL in metha-
nol and was centrifuged for 5 min at 6600 rpm and loaded
for analysis. A reverse phase Luna C18 column (60  3 mm,
3 lm) [Phenomenex, Torrance CA, US], was used to carry out
HPLC analysis of the sample. The mobile phase consists of
water (þ0.1% formic acid) A and methanol (þ0.1% formic
acid) B at a flow rate of 360 lL/min. The gradient was config-
ured to be a linear gradient from 96% A to 100% B over
14 min, followed by 100% B for 4 min, then a return to the
initial concentration of 96% A in 0.6 min, and allowed to
equilibrate for 4.6 min. The column oven condition was kept
at 30  C, and the injection volume was 6 lL. Spectrometry
analysis was carried out in positive mode with a nominal
mass resolving power of 60,000 at 400 m/z, spray voltage of
6 kV, and a scan rate of 1 Hz. The spectrometer was run with
a capillary temperature of (300  C), a tube lens of 100 V, colli-
sion gas was Argon and Nitrogen as sheath gas (66 arbitrary
units), and auxiliary gas (8 arbitrary units) (Bedane, Zu €hlke,
and Spiteller 2020; Adeyoju et al. 2022). Xcalibur software
2.2.48 was used for data analysis. Compounds were proposed
by comparison of acquired MS data with literature.
2.9. Protein and ligand preparation
The three-dimensional crystallographic structure of plasmepsin
II (PDBID: ILEE) and P. falciparum dihydrofolate reductase-thymi-
dylate synthase (PfDHFR-TS) (PDBID: 1J3I) were downloaded
from the protein data bank (www.rcsb.org). To obtain clean
proteins suitable for docking, the co-factors, ions and water
molecules were removed. Also, residues interacting with each
co-crystallized ligand were viewed on PyMol before the co-crys-
tallized ligand was removed to obtain a bare protein.
Thereafter, the clean proteins were saved in PDB format. The
three-dimensional conformers of secondary metabolites identi-
fied from the rhizome extract of Z. officinale were retrieved in
SDF format from the PubChem database (https://pub-chem.
JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 3
ncbi.nlm.nih.gov/). The ligands were thereafter loaded on the
open babel interface of PyRx 0.8 software, and its energy was
minimized under the MMFF94x forcefield and converted to PDB
and later to PDBQT for docking purposes (Faloye et al. 2021).
2.10. Validation of docking methodology and molecular
docking
Before molecular docking was performed, the docking pro-
cedure was validated by re-docking the co-crystallized
ligands in the binding pocket by considering residues with
5 Å (Table 1).
To perform docking studies of the identified phytochemi-
cals against the Plasmodium Plasmepsin II (PDBID: ILEE) and
P. falciparum dihydrofolate reductase-thymidylate synthase
(PfDHFR-TS) (PDBID: 1J3I) enzymes, the clean enzymes were
converted to PDBQT format using the MGL Tool. The pro-
teins were then loaded into the Autodock Vina interface of
PyRx 0.8 software (Trott and Olson 2010), and amino acid
residues within 5 Å were selected for each enzyme.
Thereafter, the grid box center and size were adjusted as
appropriate (Table 2). Then the PDBQT format of the ligand
was loaded, and docking was performed against each
enzyme at an exhaustiveness of 60. After the docking pro-
cedure was completed, the nine binding poses for each pro-
tein-ligand complex were ranked based on their RMSD
values, and the best pose with the lowest RMSD value was
considered the best prediction. The dock poses were
imported into the Discovery Studio visualizer and interac-
tions such as hydrogen bonding, pi, and hydrophobic inter-
actions were analyzed.
2.11. Molecular dynamics simulation of protein–ligand
complexes
The docked complex of promising compound and control
drug were furthermore optimized using computational
molecular dynamics simulations in the Desmond module of
the Schrodinger Maestro 2018.1 program. Firstly, a system
builder tool was used to prepare a simulation system built
with a TI3P model and a cubic box around the docked com-
plex’s system. The built system was further energy minimized
using a simulated annealing algorithm with 2000 iterations
and a 1.0 kcal/mol/Å convergence limit. Finally, the produc-
tion run was done for the 100 ns simulation time, and the
obtained trajectories were analyzed using the simulation
interaction programme (Saraf et al. 2022)
2.12. DFT studies of the hit molecule and chloroquine
DFT studies of gingerenone A (hit molecule) and chloroquine
(standard drug) were performed on Spartan 14 programme
using B3LYP functional (Lee-Yang-Parr exchange-correlation
functional method) and 6-31G basis set (Becke 1993). During
the calculations, the values of the frontier orbital energies
were computed from the most established conformation of
the compounds (See supplementary material).
4 K. O. FALOYE ET AL.
Table 1. Enzymes, predicted active residues, and RMSD values.
PDB ID Co-crystallized ligand
1LEE 4-amino-n-f4-[2-(2,6-dimethyl-phenoxy)-
acetylamino]-3-hydroxy-1-isobutyl-5-phenyl-
pentylg-benzamide
1J3I 6,6-dimethyl-1-[3-(2,4,5-trichlorophenoxy)propoxy]-
1,6-dihydro-1,3,5-triazine-2,4-diamine
Table 2. Grid box dimension of plasmepsin II and PfDHFR-TS.
Centre Size
Enzymes x y z x y z
1LEE 32.4490 33.3825 13.6370 28.0068 23.0537
1J3I 29.9029 9.2940 58.5605 25.5791 25.8596
2.12.1. ADME studies
The ADME studies of gingerenone A were performed by con-
sidering descriptors like solubility, blood-brain barrier permeabil-
ity, hepatotoxicity, human intestinal absorption, carcinogenicity
and acute oral toxicity using an online server (http://biosig.
unimelb.edu.au/pkcsm/prediction) (Awotuya et al. 2022).
3. Results and discussion
3.1. Dose-response study of the methanol extract of Z.
officinale rhizome using the 4-day test
Medicinal plants have been used from time past in the man-
agement of different diseases (Zareen et al. 2021). The plant
kingdom and the forest have been referred to as “the sleep-
ing giant of drug development” and “God’s own pharmacy”
(Treben 1986; Verpoorte et al. 1987). The forest needs to be
further exploited for useful drugs. Both developing and
developed countries utilize traditional herbal remedies in
their primary health care (Ganesan 2008). Many plants have
also been reported for their pharmacological properties
(Aye et al. 2019). This present study evaluates the in vivo
antiplasmodial activity of the methanol extract of Z. officinale
rhizome using the 4-day suppressive test. Percentage chemo-
suppression and survival time were used as parameters in
the assessment of the activity of the extract.
The percentage reduction in parasitaemia at all doses of
Z. officinale rhizome extract was significantly lower (p < 0.05)
than the negative control which gave 7.15 ± 0.92% and com-
parable to that of chloroquine (2.0005 ± 0.09%). At doses of
50, 100, 200 and 400 mg/kg BWT, the extract elicited
3.237 ± 0.37, 2.8445 ± 0.17, 2.483 ± 0.13 and 3.704 ± 0.56% in
parasitaemia, respectively.
The extract of Z. officinale gave the highest % chemosup-
pression of 63.64 ± 4.73% elicited at 200 mg/kg was fairly
comparable to that of chloroquine (Table 3). This result was
comparable to the chemosuppressive activity of the extract
at 50 mg/kg and 100 mg/kg. This result was significantly
higher than the value of 32.83 ± 1.03% at 1000 mg/kg, which
was reported for Z. officinale root extract by Biruksew et al.
(2018). The result was also similar to the earlier results
reported on the in vivo antimalarial activities of the stem
Active site residues within 5 Å RMSD value
Ile32, Asp34, Gly36, Ser37, Ala38, Met75, Asn76, Tyr77, 1.98
Val78, Ser79, Phe111, Phe120, Ile123, Leu131, Ile133,
Tyr192, Ile212, Asp214, Gly216, Thr217, Ser218,
Ala219, Thr221, Ile290, Leu292, Phe294, Ile300
Ile14, Cys15, Ala16, Leu46, Trp48, Lys49, Asp54, Met55, 0.96
Tyr57, Phe58, Ser108, Ser111, Ile112, Pro113, Phe116,
Leu119, Ile164, Gly165, Tyr170, Thr187
bark of Plumeria alba Linn (Apocynaceae) cultivated in
Nigeria (Adesida, Odediran, and Elujoba 2021).
The average survival time elicited by Z. officinale rhizome
24.1060
extract at all tested doses was comparable to that of the
23.0218 negative control and significantly lower than that of chloro-
quine, the positive control. It has been reported that the sur-
vival of experimental animals beyond 12 days of malarial
infection is regarded as an activity (Ajaiyeoba et al. 2006). It
has also been reported that the survival time for treated
mice in antiplasmodial studies which doubled that of the
negative control, was evidence of good antiplasmodial activ-
ities of the extract (Mukherjee 2008; Odediran, Elujoba, and
Adebajo 2014). Hence, in this present study, the near-doubled
survival time of 12 days given by the extract at 50 mg/kg com-
pared with the negative control (6.5 days) could be considered
as an indication of good antiplasmodial activity. Moreover, the
extract at 100 mg/kg did not elongate the life span of the mice
beyond that of the negative control.
The median effective doses (ED50 and ED90) are the
respective doses that will reduce the parasitaemia levels of
the untreated mice by 50 and 90%, respectively, under
standard experimental conditions (McNaught and Wilkinson
1997). Both values have been used to describe and rank the
relative antiplasmodial activities of crude extracts, partitioned
fractions and isolated chemical constituents of medicinal
plants (Odediran, Elujoba, and Adebajo 2014). The ED50
(164.2546 ± 6.94) and ED90 (265.0975 ± 27.21) values recorded
for Z. officinale were significantly lower than values recorded
from previous studies on Plumeria alba with ED50 and ED90
of 305.82 ± 9.99 and 389.74 ± 29.59, respectively (Adesida,
Odediran, and Elujoba 2021), and on Artocarpus altilis with
ED50 and ED90 values of 227.17 ± 0.3 and 372.96 ± 0.3,
respectively (Aladesanmi et al. 2022).
It can be inferred that the extract elicited good antiplas-
modial activity ( 50%) at all doses except at the highest
tested dose (400 mg/kg). This is an indication that increasing
the doses of the extract had no significant effect on its che-
mosuppressive activity. On the other hand, 50 mg/kg of Z.
officinale rhizome extract was found to give optimum anti-
plasmodial activity with better survival time than other
tested doses.
Secondary metabolites from plants are known to be
responsible for the medicinal properties of plants (Sofowora
1993). Many secondary metabolites (such as phenolic and
terpene compounds), which might be responsible for the
pharmacological properties of Z. officinale have been identi-
fied (Mao et al. 2019). Some studies have reported that high
levels of parasitemia suppression might result from mecha-
nisms of biomarker actions that indirectly affect the immune
system (Hymete et al. 2005; Biruksew et al. 2018). In vitro

Table 3. Average percentage parasitaemia, percentage chemosuppression and average survival time in the in vivo chemo-
suppressive antimalarial activities of methanol extract of Z. officinale rhizome in mice.
Doses (mg/kg) % Parasitaemia ± SEM
NC 7.1455 ± 0.92a
50 3.237 ± 0.37b
100 2.8445 ± 0.17b
200 2.483 ± 0.13b
400 3.704 ± 0.56b
CQ 2.0005 ± 0.09b
ED50 164.2546 ± 6.94
ED90 265.0975 ± 27.21
Keys: Data show the mean ± SEM, n ¼ 5: NC (negative control): Tween 80 in normal saline; CQ ¼ Chloroquine (10 mg/kg).
Only values with different superscripts within columns are significantly different (p < 0.05, one-way analysis of variance fol-
lowed by the Student–Newman–Keul’s post hoc test). ED50 and ED90 are doses of the extracts that gave 50 and 90% activity,
respectively.
Figure 1. LC–MS chromatogram of secondary metabolite profiling of Z. officinale.
antiplasmodial activities of some species in the family,
Zingiberaceae have been reported (Biruksew et al. 2018),
which support the antiplasmodial effect of the methanol
extract of Z. officinale rhizome reported in this current
findings.
3.2. Profiling of secondary metabolites in the rhizome
of Z. officinale methanolic extract
An LC–MS method was developed, and the chromatogram
obtained is presented in Figure 1.
Seven compounds were tentatively identified from the
extract of Z. officinale on the basis of the molecular ions,
mass fragments and literature comparison (Table 4).
Ginger is rich in phenolic compounds (Tohma et al. 2017;
Ali, El-Nour, and Yagi 2018). Shogaols are a class of phenolic
compounds commonly found in ginger (Prasad and Tyagi
2015; Mao et al. 2019). They are formed via the conversion
of the thermally unstable gingerols (an abundant class of
phenolic compound in ginger) at high temperatures. Entries
4, 5 and 7 eluting at retention times (rt) 23.52, 25.85 and
27.62 were tentatively identified as 6-shogaol (m/z 277.1801
[M þ H]þ), 8-shogaol (m/z 305.2112 [M þ H]þ) and 10-shogaol
(m/z 333.2425 [M þ H]þ), respectively. These compounds
have been previously isolated and identified in literature
(Chen et al. 1986).
The peak at rt 16.82 min (entry 2) was tentatively identi-
fied as gingerenone A (Endo, Kanno, and Oshima 1990; Peng
et al. 2012). It showed a molecular ion at m/z 357.1698
[M þ H]þ with the formula consistent with C21H24O5. Entry 6
at rt 26.36 min was tentatively identified as the fatty acid,
linolenic acid. Linolenic acid was previously identified with
the aid of an HPLC by Lee et al. (2014). The peak at entry 3,
eluting at rt 20.89 with a molecular ion at m/z 137.0598
JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 5
% Suppression ± SEM Average survival time ± SEM
0 ± 0.00a 6.5 ± 0.96a
52.95 ± 7.46b,c 12.00 ± 0.4a
58.70 ± 4.49b,c 5.50 ± 3.18a
63.64 ± 4.73b,c 9.50 ± 0.29a
47.88 ± 6.10b 8.00 ± 1.00a
70.75 ± 3.56c 24.25 ± 2.18b
[M þ H]þ, was tentatively identified as 4-methoxybenzalde-
hyde (Zhong et al. 2022). Entry 1 (m/z 153.0547 [M þ H]þ),
consistent with the formula C8H8O3 was identified as vanillin
(Brillatz et al. 2020).
3.3. Molecular docking analysis
The molecular docking procedure validation showed that the
RMSD values calculated between the co-crystallized and re-
docked ligands were < 2 Å (Table 1). Also, the structure of
the co-crystallized and re-docked ligands were almost super-
imposed on each other, thereby confirming the correctness
of the docking procedure adopted for the study (see supple-
mentary file (Figure 1))
Molecular docking is widely used for screening and identi-
fying drug candidates from synthetic and natural sources. In
this study, the phytochemicals identified from the metha-
nolic extract of the rhizome of Z. officinale were docked
against antimalarial receptors (plasmepsin II and PfDHFR-TS).
Also, chloroquine was selected as the standard drug, and its
binding energy was set as the cut-off point to identify the
hit molecule.
Gingerenone A was selected as the hit molecule when
the phytochemicals were screened against plasmepsin II and
their binding energies were compared with chloroquine
(6.4 kcal/mol). The hit molecule elicited a considerably high
binding energy of 7.0 kcal/mol but was not as high as the
synthetic co-crystallized ligand (9.6 kcal/mol). It established
hydrophobic interactions with Met15, Ile32, Tyr77, Ile123,
and Leu131 compared to chloroquine that formed hydropho-
bic interactions with Met15, Ile32, Tyr77, Val78, Ile100, Ile123,
Leu292, and Ile300. The hydrogen atom of the hydroxyl
group on the aromatic rings of gingerenone A participated
in hydrogen bonding interaction with Asn76 at a bond
6 K. O. FALOYE ET AL.

distance of 2.18 Å, while hydrogen bonding interaction was organic compounds’ binding affinity and bioactive potential
established between chloroquine and plasmepsin II at a lon- (Chen et al. 2016; Abelian et al. 2021). Hydrogen bonds play
ger bond distance of 2.21 Å (Figure 2a and b). Furthermore, a significant role in protein-drug interactions and are com-
the stability of gingerenone A was strengthened in the bind- monly found between protein-ligand complexes (Grabowski
ing pocket of plasmepsin II by forming pi-alkyl and pi-sigma 2020). Gingerenone A formed a hydrogen bond with the
interactions with Ile32 and Tyr77, while chloroquine formed amino acid residues of plasmepsin II and PfDHFR-TS at a
pi-alkyl interactions with Tyr77 and Val78. Gingerenone A considerable bond distance that is strong enough to
and the co-crystallized ligand formed common hydrogen strengthen its direct interaction with the receptors in con-
bonding interaction with Asp76, hydrophobic interactions trast to chloroquine.
with Ile32, Tyr77, Ile123, Leu131, and pi-interactions with Hydrophobic interactions are established between the
Ile32 and Tyr77. lipophilic groups of chemical compounds and non-polar side
In the molecular docking studies of identified phytochem- chains of amino acids (Chen et al. 2016). It contributes to the
icals and chloroquine against PfDHFR-TS, gingerenone A was binding affinity of chemical compounds and provides
selected as the hit molecule. The compound elicited a bind- insights into their bioactivity potentials (Abelian et al. 2021).
ing energy of 8.0 kcal/mol which is higher than chloroquine The lipophilic atoms on gingerenone A and chloroquine
(7.2 kcal/mol) but lower than that of the co-crystallized ligand formed hydrophobic interactions with non-polar atoms of
(8.9 kcal/mol). An oxygen atom on the gingerenone A moi- the amino acid residues of the receptors’ active site, and
ety formed a hydrogen bonding interaction with Ser111 at a these interactions are good enough for the hit molecule to
bond distance of 2.62 Å, while chloroquine also formed elicit considerably high binding energy than the latter and
hydrogen bonding interaction with Ile164 at a bond distance also reveal it as a good inhibitor against the receptors. The pi-
of 3.02 Å. Gingerenone A also established hydrophobic inter- interactions are established when pi-electron cloud establishes
action with Ala16, Leu40, Leu46, and Phe58, while chloro- interaction between the aromatic group and pi-electron of phy-
quine had five hydrophobic interactions with Cys15, Leu40, tochemicals to facilitate their stability in the receptor’s binding
Phe58, Ile112, Leu119, and Ile164. Furthermore, pi-alkyl inter- site (Mousavi et al. 2021). Both gingerenone A and chloroquine
action was observed between gingerenone A-Leu40 and have aromatic ring systems and formed pi-interaction at the
chloroquine-Phe58 and Ile164 (Figure 3a and b). The hit mol- receptors’ binding sites.
ecule established only two hydrophobic interactions with A similar study conducted on quercetin against plasmepsin
Ile112 and Leu119. II showed that gingerenone elicited better binding energy than
Non-covalent interactions and chemical bonding is the the former which gave 6.36 kcal/mol (Hasan et al. 2022). In
major route through which chemical compounds binds to another study conducted by Olanlokun et al. (2019) whereby a
receptors (Grabowski 2020). Hydrogen bonding and hydro-
novel phytochemical (tetrahydro-4-((E)-7-hydroxy-10-methoxy-6,
phobic interactions are major determinants in evaluating
14-dimethyl-15-m-tolylpentadec-13-enyl) pyran-2-one) was
screened against plasmepsin II, a binding energy comparable
Table 4. Secondary metabolite profiling of extract of Z. officinale.
to gingerenone A was obtained. Also, Kyei et al. (2022)
m/z [Adduct]
Entry Rt (min) [M þ H]þ Formula Name screened pyrimethamine against PfDHFR-TS and obtained a
1 10.96 153.0547 C8H8O3 Vanillin binding energy of 7.8 kcal/mol which is lower than that of
2 16.82 357.1698 C21H24O5 Gingerenone A gingerenone A. Also, the binding energy of gingerenone A
3 20.89 137.0598 C8H9O2 4-Methoxybenzaldehyde against PfDHFR-TS is higher than values reported for lascu-
4 23.52 277.1801 C17H24O3 6-Shogaol
5 25.85 305.2112 C19H28O3 8-Shogaol floxacin (6.59 kcal/mol) and moxifloxacin (5.65 kcal/mol)
6 26.36 279.2320 C18H30O2 a-Linolenic acid (Balogun, Omoboyowa, and Saibu 2020). Gingerenone A has
7 27.62 333.2425 C21H32O3 10-Shogaol
previously been reported to possess interesting biological

Figure 2. Interaction diagrams of the docked complexes of (a) ILEE-gingerenone A and (b) ILEE-chloroquine.

Figure 3. Interaction diagrams of the docked complexes of (a) IJ3I-gingerenone A and (b) IJ3I-chloroquine.
activities, such that it attenuated TNF-a, LPS-induced mono-
cyte-endothelial adhesion, and inhibited Staphylococcus aur-
eus SaHPPK in silico (Kim et al. 2018; Rampogu et al. 2018).
Other studies revealed gingerenone A as an excellent thera-
peutic agent with strong antimicrobial, anticancer and anti-
diabetic activities (Chen et al. 2018; Rampogu et al. 2018; Yu
et al. 2022). The binding energy obtained for gingerenone A
as compared to chloroquine in the molecular docking studies
against the malaria enzymes is a strong indication that the
hit molecule may be responsible for the antimalarial activity
elicited by the methanolic extract of Z. officinale.
3.4. Molecular dynamics simulation analysis
Molecular dynamics simulation is a vital study to investigate
the structural dynamics, conformation, interactions and sta-
bility of protein-ligand complexes (Yadav, Tripathi, and Yadav
2021; Iqbal et al. 2022). In this study, the 1LEE-gingerenone
A and 1J3I-gingerenone A complexes were subjected to a
100 ns simulation study to gain better insight into any
change in the protein conformational study that may arise
from the gingerenone A binding. The stability of gingere-
none A in 1LEE-gingerenone and 1J3I-gingerenone A com-
plexes was estimated by considering their RMSD (root mean
square deviation) and RMSF (root mean square fluctuations).
The protein RMSD plot of the Ca backbone (blue colour)
of 1LEE and reference frame backbone obtained for the
100 ns simulation is presented in Figure 4. The RMSD plot
showed an initial increase from 0 to 5 ns followed by a stable
system from 6 to 60 ns and a slope that ranged between 1.7
and 2.4 Å, while the Ca backbone of chloroquine-1LEE com-
plex was stable up to 63 ns followed by a slight deviation
and stability was observed to 100 ns. In the ligand RMSD
plot for the gingerenone A fitted on 1LEE backbone, serial
fluctuation was observed between 0 and 40 ns followed by a
stable system 41–90 ns and a sharp increase that range
between 9 and13 Å, while chloroquine was stable up to
28 ns followed by consistent deviation to 100 ns (Figure 4a
and b). The RMSF plot of gingerenone A-1LEE showed that
JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 7
the protein fluctuated slightly between 0.6 and 3.4 Å, while
that of chloroquine-1LEE ranged between 0.5 and 3.2 Å in
the 100 ns gingerenone A-ILEE simulation period (Figure 4c
and d). Also, the contact plot showed good hydrogen bonds,
hydrophobic interactions and water bridge. Gingerenone A
participated in hydrogen bonding interaction with Ala38,
Asn39, Val78, Ser79, Thr114, and Ile133, while hydrophobic
interaction was observed with Tyr41, Tyr77, Phe111, Ile123,
and Ile133. Furthermore, a water bridge was formed with
Asp34, Val78 and Ser79 (Figure 4e). Comparing the contact
plot of chloroquine with gingerenone A, chloroquine had
hydrogen bonding interaction with Asp34, Gln142, Asp162,
hydrophobic interactions with Tyr77, Phe111, Phe241 and
water bridge with Asp214 (Figure 4f). Overall, the RMSD plot
showed that gingerenone A was more stable than chloro-
quine in the binding pocket of plasmepsin II. Also, common
hydrophobic interactions were established with Tyr77 and
Phe111.
The 1J31-gingerenone A complex RMSD increased for
about 10 ns followed by stability between 1.6 and 2.6 Å
throughout the simulation period. Also, the ligand RMSD for
the gingerenone A fitted on the 1J3I backbone showed ini-
tial stability from 0 to 9 ns followed by a sharp increase to
15 ns. Stability was observed between 16 and 43 ns followed
by a sharp increase and fluctuation between 44 and 55 ns
and stability for the remaining simulation period, while initial
deviation was observed for chloroquine and the system was
stable till 90 ns followed by a slight deviation to 100 ns
(Figure 5a and b). In the RMSF plot, slight fluctuation was
observed with the protein around 0.6–4.8 Å, while fluctua-
tions were observed for the protein in the chloroquine-1J3I
simulation (Figure 5c and d).
Furthermore, the protein–ligand interaction chart showed
the presence of hydrogen bonds, hydrophobic interactions,
and water bridge. In the contact plot, gingerenone A estab-
lished hydrogen bonding interaction with Ala16, Cys50,
Asp54, Ser111, and Tyr185, while hydrophobic interaction
was observed between the phytochemical and Ala16, Leu40,
Leu46, Met55, Phe58, Ile112, and Pro113. Additionally, water
8 K. O. FALOYE ET AL.
Figure 4. Illustration diagram of (a) RMSD of ILEE-gingerenone A, (b) RMSD of ILEE-chloroquine, (c) RMSF of ILEE-gingerenone A, (d) RMSF of ILEE-chloroquine, (e)
contact plot of ILEE-gingerenone A and (f) contact plot of ILEE-chloroquine.
bridge interaction was formed with Gly44, Tyr48, Lys49,
Cys50, Asp54, Thr107, Ser111, Arg122, and Ile164 (Figure 5e),
while chloroquine established interactions with fewer amino
acids (hydrogen bonding interactions with Asp54, hydropho-
bic interactions with Phe58, Leu46 and water bridge with
Ile164) (Figure 5f).
The RMSD and RMSF plots help to understand the flexibil-
ity of amino acid residues and the stability of ligands in the
protein’s binding pocket (Mousavi et al. 2021). Also, interac-
tions like hydrogen bonding, hydrophobic, and water bridges
contribute to the stability and binding energy elicited by
ligands (Yadav, Tripathi, and Yadav 2021). Gingerenone A
was more stable than chloroquine and established good
interactions with the residues at the proteins’ active sites
and also corroborated the findings in the molecular docking
studies.
3.5. MMGBSA analysis
The MMGBSA is a less expensive computational method of
estimating the binding affinity of docked protein-ligand com-
plexes compared to thermodynamic integration and free
energy perturbation methods (Iqbal et al. 2022).
In this study, the MMGBSA results showed that gingere-
none A had good binding free energy with plasmepsin II
(37.07 kcal/mol) and PfDHFR-TS (-49.09 kcal/mol) (Table 5).
Also, the covalent, Van der Waal, solvation, and coulomb
energy value computed for the ILEE-gingerenone A and 1J3I-

Figure 4. Continued.
gingerenone A complex contributed to the binding and sta-
bility of the ligand throughout the simulation period.
3.6. Frontier molecular orbital analysis of the hit
molecule and standard drug
The frontier molecular orbital analysis of phytochemicals
gives useful information about their drug candidacy through
the estimation of their physiochemical properties like highest
occupied molecular orbital (HOMO), lowest unoccupied
molecular orbital (LUMO), energy gap, hardness, softness,
electrophilicity index, and electronegativity.
The EHOMO of a chemical compound is a measure of its
electron donating potential while the ELUMO is used to meas-
ure its electron accepting property. Therefore, the higher the
EHOMO value, the greater the electron donating tendency of
the molecule, while a higher ELUMO value indicates a higher
electron accepting tendency (Bhavani et al. 2015). In this
JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 9
study, chloroquine has a higher EHOMO value (5.49 eV) as
compared to gingerenone A (5.35 eV), indicating that
chloroquine has a greater electron donating property than
gingerenone A. Conversely, gingerenone A (1.50 eV) has a
better electron withdrawing property than chloroquine
(1.04 eV). The EHOMO and ELUMO values obtained were as a
result of the electric charges delocalized over the p-network
and aromatic ring system of gingerenone A and chloroquine
(Janjua et al. 2014).
The energy gap of a molecule is the excitation energy
required to cause an electron transfer. It is used to study the
chemical reactivity, stability, and bioactive properties of sec-
ondary metabolites (Subramanian, Sundaraganesan, and
Jayabharathi 2010; Obi-Egbedi et al. 2011). Chloroquine
(4.45 eV) has a higher energy gap as compared to gingere-
none A (3.85 eV), signifying that chloroquine is more stable
but less reactive, at the active site of the antimalarial
enzymes (Figure 6). Also, the decreased energy gap of
10 K. O. FALOYE ET AL.
Figure 5. Illustration diagram of (a) RMSD of 1J3I-gingerenone A, (b) RMSD of 1J3I-chloroquine, (c) RMSF of 1J3I-gingerenone A, (d) RMSF of 1J3I-chloroquine, (e)
contact plot of 1J3I-gingerenone A and (f) contact plot of 1J3I-chloroquine.
gingerenone A indicates that it will undergo easy excitation
of electrons from the ground state (Raheem, Al-Shejyri, and
Al-Bermany 2012).
The chemical hardness and softness of chemical com-
pounds are important in accurately predicting their stability
and reactivity (Ayeni et al. 2020). Chemical compounds with
a lower hardness value have a better tendency to exchange
charges at the binding pocket of enzymes (Bulbul et al.
2021; Owoseeni et al. 2022). Therefore, they are essential
electronic parameters in predicting the pharmacological
potential of secondary metabolites. Gingerenone A (1.93 eV;
0.52 eV) has close hardness and softness values to
chloroquine (2.23 eV; 0.45 eV). Hence, it could be said that
gingerenone is a promising antimalarial agent. Additionally,
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 11

Figure 5. Continued.
12 K. O. FALOYE ET AL.

chemical potential helps to predict the reactivity and stability 3.7. ADMET prediction
of secondary metabolites. The higher the chemical potential
The ADMET properties of gingerenone A were evaluated to
of a molecule, the higher its reactivity and the lower its sta-
understand its metabolism and safety when taken by malaria
bility (Subramanian et al. 2010; Awotuya et al. 2022).
subjects. The solubility and human intestinal absorption
The electronegativity and electrophilicity index of chloro-
property of a drug candidate provide information on the
quine and gingerenone A were also calculated. The electro-
ease of formulation, mode of administration and rate of
negativity of a chemical compound is a measure of its
absorption (Ogboye et al. 2022). Blood-brain barrier (BBB)
electron withdrawing property. It helps in evaluating the bio-
permeability property of a drug candidate measures its abil-
logical property of secondary metabolites (Domingo et al.
ity to cross the BBB layer without any hindrance and it is
2016; Hussein and Azeez 2021). Gingerenone A (3.44 eV) has
important for central nervous system (CNS) drug design
a better electron withdrawing property due to the presence
(Sharma et al. 2020). The carcinogenicity property of a drug
of more electronegative elements as compared to chloro-
candidate elucidates its ability to either speed up the forma-
quine (3.27 eV). The electrophilicity index is directly related
tion of cancerous cells or not, while hepatotoxicity study
to the binding affinity elicited by a secondary metabolite at
helps to predict the possible toxicity of the chemical com-
the active site of a receptor. It also measures the stability of
pound to the liver (Owoseeni et al. 2022). The acute oral tox-
a chemical compound when it obtains more electron density
icity studies of a drug candidate provide information about
from its environment (Domingo et al. 2016). A strong electro-
its safety when taken orally by malarial subjects.
phile possesses a high electrophilicity index value and high
In this study, gingerenone A gave a solubility value of
binding energy against a receptor (Mumit et al. 2020;
4.66 and a human intestinal absorption value of 93%. A
Olajubutu et al. 2022). Gingerenone A (3.16 eV) had a higher
drug candidate with good absorptivity, when administered,
electrophilicity index than chloroquine (2.39 eV) which also
must have a solubility value ranging between 6.5 and 0.5,
corroborated the binding energy elicited by the hit molecule
while human intestinal absorption should be more than 30%
in the molecular docking and MMGBSA studies.
(Sharma et al. 2020; Awotuya et al. 2022). The hit molecule
Table 5. MMGBSA DG binding scores of the top-ranked molecules.
also showed good a BBB permeability property, signifying
that it is capable of acting as a good CNS drug. In terms of
DG bind
DG bind DG bind solvation DG bind toxicity, gingerenone A is a non-carcinogenic compound and
P-L complex DG bind coulomb covalent energy vdW not toxic to the liver. The oral acute and chronic toxicity
1LEE-gingerenone A 37.07 24.98 8.05 41.78 38.10 studies showed that gingerenone A is not toxic when admin-
1J3I-gingerenone A 49.09 8.84 7.20 33.53 52.51
istered orally to malaria subjects. The overall toxicity profile

Figure 6. Illustration diagram of the HOMO and LUMO of (a) gingerenone A and (b) chloroquine.

of the hit molecule shows it is safe for further clinical and
pharmacokinetic investigations.
4. Conclusion
This study evaluated the antimalarial activity of the methanolic
extract of Z. officinale rhizome, identified the phytoconstituents
in the extract, performed molecular docking and molecular
dynamics simulation studies against plasmepsin II and PfDHFR-
TS receptors to identify the hit molecule that may be respon-
sible for the antimalarial activity. It also carried out density
functional theory calculations to obtain the electronic properties
of the hit molecule and predicted the ADMET profile to esti-
mate the safety and drug candidacy of the top-ranked phyto-
chemical. The results obtained showed that the extract elicited
a dose-dependent chemosuppressive activity up to 200 mg/kg.
At the lowest dose of 50 mg/kg, the extract gave antiplasmodial
activity >50% and the highest survival time of 12 days. The
highest antimalarial activity of 63% elicited by the methanolic
extract at 200 mg/kg confirms the usage of Z. officinale rhizome
in ethnomedicine. The LC-MS secondary metabolite profiling
identified vanillin, gingerenone A, 4-methoxybenzaldehyde, 6-
shagaol, 8-shogaol, 10-shogaol and a-linolenic acid as the
chemical constituents present in the methanolic extract of the
Z. officinale rhizome. The molecular docking study identified
gingerenone A as the hit molecule against plasmepsin II and
PfDHFR-TS with a binding energy higher than that of chloro-
quine. Gingerenone A established good hydrophobic, hydrogen
bonding and pi-interactions which contributed significantly to
its good binding energy and stability at the enzymes’ binding
pocket. The RMSD plots obtained in the 100 ns molecular
dynamics simulation showed that gingerenone A exhibited bet-
ter stability at the binding pocket of plasmepsin II and PfDHFR-
TS receptors, while the RMSF plots suggested slight fluctuations
were observed with the amino acid residues of the malaria
enzymes. The contact plots showed that gingerenone A con-
solidated its stability during the 100 ns molecular dynamics
simulation by forming water bridge, hydrogen bonding and
hydrophobic interactions. Also, the electrophilicity index and
HOMO-LUMO energy gap values obtained from density func-
tional theory calculations for gingerenone A supported the
binding energy elicited by gingerenone A against plasmepsin II
and PfDHFR-TS enzymes. In addition, the toxicity profile gener-
ated from the ADMET profile showed that gingerenone A has
good solubility, human intestinal absorption properties and safe
for malaria subjects. The isolation and extensive antimalarial
studies of gingerenone A is thereby recommended.
Acknowledgement
S.A.O, K.O.F, U.I.O, I.I.O and O.D.D are thankful to Ms. Eva Wieczorek
(Institute of food and environmental research, Technical University of
Dortmund) for her assistance with LC-MS data acquisition.
Disclosure statement
The authors declare that they have no conflicts of interest with the con-
tents of this article.
JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 13
Funding
The authors did not receive any external funding.
ORCID
Adetola H. Adewole http://orcid.org/0000-0003-4983-245X
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