Antidepressant-like effect of dehydrozingerone from Zingiber officinale by elevating monoamines in brain: in silico and in vivo studies

Article abstract of DOI:10.1007/s43440-021-00252-0

Background: Dehydrozingerone (DHZ) is an active ingredient of Zingiber officinale and structural half analogue of curcumin. In the present study, DHZ was evaluated for monoamine oxidase (MAO) inhibitory activity in silico and antidepressant activity in vivo. Method: The?binding affinity of DHZ?with MAO-A (PDB ID: 2Z5Y) was assessed?using Schrodinger's Maestro followed by free energy calculation, pharmacokinetic property prediction using Qikprop and Molecular dynamics simulation using Desmond. In vivo antidepressant activity of DHZ was evaluated on C57 BL/6 male mice using Escilatopram as the?standard?antidepressant. Open field test (OFT), forced swimming test (FST) and tail suspension test (TST) were used to evaluate the antidepressant effect of the drugs on days 1 and 7. Following?the behavioural study, neurotransmitters (noradrenaline, dopamine and serotonin) were estimated using liquid chromatography–mass spectrometry. Results: DHZ demonstrated a greater binding affinity for the MAO-A enzyme compared to moclobemide?in silico. Immobility in TST and FST were significantly (p?

Full text of DOI:10.1007/s43440-021-00252-0

Pharmacological Reports
https://doi.org/10.1007/s43440-021-00252-0
ARTICLE
Antidepressant‑like efect of dehydrozingerone from Zingiber
ofcinale by elevating monoamines in brain: in silico and in vivo
studies
Sudheer Moorkoth1 · N. Sai Prathyusha1 · Suman Manandhar2 · Yuanxin Xue3 · Runali Sankhe2 · K. S. R. Pai2 ·
Nitesh Kumar2,4
Received: 20 October 2020 / Revised: 12 March 2021 / Accepted: 16 March 2021
© The Author(s) 2021
Abstract
Background Dehydrozingerone (DHZ) is an active ingredient of Zingiber ofcinale and structural half analogue of curcumin.
In the present study, DHZ was evaluated for monoamine oxidase (MAO) inhibitory activity in silico and antidepressant
activity in vivo.
Method The binding afnity of DHZ with MAO-A (PDB ID: 2Z5Y) was assessed using Schrodinger’s Maestro followed
by free energy calculation, pharmacokinetic property prediction using Qikprop and Molecular dynamics simulation using
Desmond. In vivo antidepressant activity of DHZ was evaluated on C57 BL/6 male mice using Escilatopram as the stand-
ard antidepressant. Open feld test (OFT), forced swimming test (FST) and tail suspension test (TST) were used to evaluate
the antidepressant efect of the drugs on days 1 and 7. Following the behavioural study, neurotransmitters (noradrenaline,
dopamine and serotonin) were estimated using liquid chromatography–mass spectrometry.
Results DHZ demonstrated a greater binding afnity for the MAO-A enzyme compared to moclobemide in silico. Immobility
in TST and FST were signifcantly (p < 0.05) reduced in vivo with 100mg/kg DHZ as compared to respective controls. DHZ
treatment was more efective 1 h post treatment compared to vehicle control. A signifcant increase in levels of neurotrans-
mitters was observed in mice brain homogenate in response to DHZ treatment, reassuring its antidepressant-like potential.
Conclusion DHZ demonstrated MAO-A inhibition in silico, and the increased neurotransmitter levels in the brain in vivo
were associated with an antidepressant-like efect.
Keywords Dehydrozingerone · Depression · Monoamine oxidase-A · Neurotransmitter · Molecular dynamics · LCMS
Introduction
Sudheer Moorkoth, N. Sai Prathyusha and Suman Manandhar
contributed equally.
Depression is the most prevalent mental disorder world-
wide afecting more than 300 million people of all ages.
The chronic and severe form of depression often leads to sui-
cidal tendencies, with approximately 800,000 people com-
mitting suicide each year, primarily within the age group
of 15–29 years old. The serendipitous discovery of the frst
monoamine oxidase (MAO) inhibitor occurred after observ-
ing the side efects of an antitubercular drug in the 1950s,
Iproniazid, which induced euphoria, sleep improvement and
psychostimulation [1]. The role of several neurotransmitters
such as serotonin, norepinephrine/epinephrine and dopamine
has been indicated in depression [2, 3]. Currently, available
antidepressants drugs (tricyclic and tetracyclic antidepres-
sants, MAO inhibitors, selective serotonin and noradrena-
line reuptake inhibitors and mood stabilizers) are prescribed
* Nitesh Kumar
niteshkumar43@gmail.com;
nitesh.kumar04@niperhajipur.ac.in
1
Department of Pharmaceutical Quality Assurance, Manipal
College of Pharmaceutical Sciences, Manipal Academy
of Higher Education, Manipal, Karnataka 576104, India
2
Department of Pharmacology, Manipal College
of Pharmaceutical Sciences, Manipal Academy of Higher
Education, Manipal, Karnataka 576104, India
3
Faculty of Health Sciences, McMaster University, Hamilton,
Ontario, Canada
4
Department of Pharmacology and Toxicology, National
Institute of Pharmaceutical Education and Research, Hajipur,
Bihar 844102, India
Vol.:(0123456789)
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S. Moorkoth et al.
based on the medical condition and preferences [4]. The
use of tricyclic antidepressants and monoamine oxidase
inhibitors have shown short term reversal of the symptoms
of depression. Currently available antidepressants have low
efcacy and dietary restrictions, and some patients are non-
responsive altogether [5]. There also exist numerous side
efects including liver toxicity and hypertension [6].
and dopaminergic system [20]. Curcumin was found to have
potent inhibitory activity for both MAO-A and MAO-B
enzymes [21]. DHZ has been reported to have potent anti-
depressant activity by in vivo study through the involvement
of serotonergic and noradrenergic systems [22]. Antidepres-
sant activity of the hydroalcoholic extract from the rhizomes
of Zingiber ofcinale has been proven. This study aims to
evaluate the involvement of MAO-A inhibitory activity of
DHZ for its antidepressant activity by computational based
in silico molecular docking and molecular dynamics simula-
tion study followed by in vivo rodent model and LC–MS-
based estimation of neurotransmitter level.
MAO, a favin adenine dinucleotide present in the mito-
chondrial outer membrane, is involved in the oxidative
deamination of monoamines including serotonin, dopa-
mine, epinephrine, norepinephrine, melatonin, etc. MAO
has two subtypes, MAO-A and MAO-B, whose amino acid
sequences are up to 70% identical, although each enzyme
has a unique substrate and inhibitor specifcities [7]. MAO
inhibitors are clinically used as therapeutics for depression,
Parkinson’s and Alzheimer’s disease. It stops the break-
down of these monoamine neurotransmitters resulting in an
increased concentration and availability [8].
Materials and methods
In silico docking study
MAO inhibitors are one of the important therapeutic
options for depression. However, due to the adverse efect
and serious drug and food interactions with the MAO inhibi-
tor, their use has been reduced. Natural compounds have
great potential for drug design and discovery for several dis-
ease conditions without the risk of several adverse efects.
Dehydrozingerone (DHZ) (4-hydroxy-3-methoxybenzalace-
tone), is the phenolic compound extracted from the rhizome
of Zingiber ofcinale having potent antioxidant, tyrosinase
inhibitory, anti-infammatory, antidepressant, antifungal and
anticancer activities [9, 10]. Ginger is one of the impor-
tant plants traditionally used since the ancient period for its
medicinal property especially in Unani, Ayurveda and Chi-
nese medicinal systems [11]. Traditional Chinese Medicinal
decoction prescribed since 1000 years for the treatment of
depression, which consists of fve indigenous herbs namely,
Pinellia ternata (Thunb.) Breit, Magnolia ofcinalis Rehd.
et Wils., Poria cocos (Schw.) Wolf, Perilla frutescens (L.)
Britt. and Zingiber ofcinale Rosc [11–13]. Ginger rhizome
has been traditionally used as one of the components in the
herbal formula Banxia houpu decoction for the treatment of
mental disorders including depression [14]. Dehydrozinge-
rone is the phenolic compound isolated from the rhizome of
Zingiber ofcinale [9]. It is the half-structural analogue of
curcumin which can be synthesized by the aldol condensa-
tion reaction between vanillin and acetone in the laboratory.
DHZ has been reported to possess cytotoxic, [15] antimi-
crobial activity, antioxidant property, antifungal activity
[16], anti-infammatory activity [17]. Antithrombotic activ-
ity of DHZ has been done in the endothelial cells show-
ing its potential as a therapeutic agent in atherosclerosis.
Antiprostate cancer property of DHZ has been proven both
in vitro as well as in vivo [18]. The antidepressant activity
of curcumin has been widely studied and has been linked to
the MAO inhibitory activity [19], through the serotonergic
The computational study and analysis were carried out using
Maestro 2018–3 molecular modelling platform by Schrod-
inger on a Linux operating system, Intel core processor with
8 GB RAM, utilizing tools like Ligprep, Protein prepara-
tion wizard, GLIDE (Grid-based Ligand Docking with Ener-
getic), Prime, Desmond and Qikprop.
Ligand and protein preparation
The 2D SDF format of DHZ and standard antidepressant
moclobemide (MAO-A inhibitor) was obtained from the
ZINC database. DHZ was prepared for docking analy-
sis using the ‘LigPrep’ tool wherein the conversion to 3D
form and generation of possible tautomers and conformers
were completed. Ligand preparation was completed at neu-
tral pH 7 2 using Epik, and energy minimization of the
ligand was under OPLS3e (Optimized potentials for liquid
simulations_3e) force feld [23].
The inhibitor bound structure of MAO-A (PDB ID:
2Z5Y) with a resolution of 2.17 Å was retrieved from RCSB
Protein Data Bank [7] and prepared using the ‘Protein prepa-
ration Wizard’ tool. It involves three steps including viz.
import and processing; review and modify; and refnement
of protein. Initially, the protein was pre-processed by the
addition of hydrogen atoms, removal of unwanted water
molecules beyond 5 Å from the hetero group and addition
of missing side chains and loops. The pre-processed protein
was then refned by hydrogen bond assignment at neutral
pH using PROPKA and energy minimization using OPLS3e
force feld.
Molecular docking
The grid was generated in the protein using the receptor
grid generation tool of the GLIDE module, considering the
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Antidepressant‑like efect of dehydrozingerone from Zingiber ofcinale by elevating…
protein-bound inhibitor. GLIDE involves a rapid, accurate
and systematic search for the conformational, positional
and orientational space followed by docking and scoring
[24]. The prepared ligands were then docked to the active
site of MAO-A, using the ’Ligand docking’ tool in the
GLIDE module with the XP (Extra precision) mode of
docking, known as rigid docking. Only the hydrogen bonds
(H-bond) formed between acceptor–donor atoms with a dis-
tance less than 3.3 Å were considered for this study. Compu-
tationally, XP docking is the more extensive mode of dock-
ing which uses the advanced scoring function. In addition,
chances of getting false-positive results are less compared
to high-throughput virtual screening and standard precision
docking mode.
receptor–ligand complex in the water box was minimized.
(3) Molecular dynamics: the trajectory of the simulation was
recorded every 30 ps, and approximately 1000 frames were
recorded for 30 ns simulation period. The root mean square
deviation (RMSD) was calculated for DHZ.
In vivo study
Experimental animals
C57BL/6 male mice of 4 months age weighing 25–30 g were
obtained from the Central Animal Research Facility (CARF)
of Manipal Academy of Higher Education, Manipal (Karna-
taka, India). Animals were acclimatized for one week in a
controlled condition of temperature (25 2 °C) with 12 h of
light and 12 h of dark cycles in laboratory conditions. Before
two hours of drug administration, the animals were kept fast-
ing to increase drug absorption. Prior approval was obtained
from Institutional Animals Ethics Committee (IAEC), Kas-
turba Medical College, Manipal Academy of Higher Edu-
cation; IAEC number: Reg No.94/PO/ReBi/S/99/CPCSEA
Dated 28/04/2017 and the experimental procedures were
done as per the CPCSEA guidelines.
The validation of the generated grid was done by the re-
docking of the known co-crystallized ligands present in the
same protein (PDB ID: 2Z5Y). RMSD value was measured
to check the suitability of the generated grid and docking
results [25].
Drug‑likeness evaluation
The ’QikProp’ module was used to study the absorption,
distribution, metabolism and excretion/toxicity properties
of DHZ. The physiochemical properties including molecular
weight (Mol wt), QPlogS (solubility), QPlog HERG (ability
to block K+ channel), QPPCaco (Caco2 cell permeability),
QPlogBBB (Blood/brain partition coefcient), human oral
absorption (% oral absorption) and rule of fve were evalu-
ated to provide a basic understanding of the pharmacokinetic
property of the molecule and predict the drug-likeness of
the molecule.
Drugs and chemicals
Noradrenaline (Sigma Aldrich > 98% purity), Serotonin
hydrochloride (Sigma Aldrich > 98% purity) and Dopa-
mine hydrochloride (Sigma Aldrich > 98.5% purity) were
procured from Durga Laboratories, Mangalore. Acetonitrile
(LC–MS grade Biosolve) was purchased from Ultra Inter-
national Lab, Bangalore. Formic acid, ethanol and sodium
carboxymethylcellulose were obtained from HiMedia Labo-
ratories Pvt. Ltd., Mumbai, India.
Molecular dynamics (MD) simulation
Drugs and proteins are surrounded by water molecules
inside the body and are continuously in motion. MD simu-
lation helps us understand a similar situation by mimick-
ing physiological condition. MD simulation study further
confrms the binding of the ligands and the stability of the
complex in simulated physiological conditions. For a bet-
ter understanding of the binding afnity and stability of
DHZ with MAO-A, MD simulation study was performed
using the ‘Desmond’ module. Three steps are involved in
MD simulation, (1) System builder: The protein–ligand
complex system was built using simple point charged water
model (SPC) in the orthorhombic water box of dimension
10 Å × 10 Å × 10 Å and the system was neutralized either
using Na+ or Cl− ions as per the requirement. NPT (Nor-
mal Pressure and Temperature) simulation system was used,
and the temperature and pressure of the system were main-
tained at 300 K and 1 bar, respectively, during the simulation
study. (2) Minimization: Before running the simulation, the
Synthesis of DHZ
DHZ was synthesized using the method as described by
Agrawal et al., [26] with a slight modifcation to increase
the yield. 2.5 g of vanillin and 20 ml of acetone were stirred
with a magnetic stirrer for 25 min. 15 ml sodium hydroxide
solution (0.25 M) was added and stirred continuously for
another two hours. Ice was added to this solution followed
by the addition of 10% HCl with continuous stirring until
a yellow precipitate formed. The precipitate was then fl-
tered and dried for 24 h. Recrystallization was done with
hot ethanol. The fnal product was characterised by mass
and infrared spectroscopy.
Treatment schedule
Animals were allocated to fve groups (n= 6 per group),
namely normal/vehicle control, escitalopram (10 mg/kg,
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S. Moorkoth et al.
po) and DHZ (100 mg/kg po) for three time points (1 h, 3 h
and 6 h groups). Dose for escitalopram and DHZ were based
on the previous reports [26, 27]. All doses were freshly pre-
pared in 0.25% CMC and animals were given treatment for
seven days. Behavioural tests were conducted on the frst and
seventh day after 1 h, 3 h and 6 h of dosing. The behavioural
studies were performed consecutively in the following order
with a gap of 1 min between each test: open feld test (OFT),
forced swim test (FST) and tail suspension test (TST).
and internal standards (ISTD, Isoprenaline) were added. In
order to extract noradrenaline, dopamine and serotonin from
the matrix, formic acid with acetonitrile and water were
added to homogenize the brain, which was then centrifuged
again for 10 min at 12,000 RPM. Then, the supernatant was
evaporated to dryness at 45 °C for 1 h using a nitrogen evap-
orator [34]. Finally, the residue was dried and reconstituted
with 200 µl of the mobile phase.
LC–MS method optimization A Thermo Scientifc (MA,
USA) UHPLC–MS with Dionex Ultimate 3000 liquid chro-
matograph interfaced with a linear ion-trap analyzer and an
electron spray ionization source was used. MS and chroma-
tographic method development were performed using LTQ
XL (MA, USA) and Chromeleon (MA, USA) softwares,
respectively. Batch analysis was done using the Xcalibur
software (MA, USA) and quantifcation was done using LC
Quan software (MA, USA).
Behavioural tests
Forced swim test (FST) After 1 h, 3 h and 6 h following
the treatment of DHZ, vehicle or escitalopram, mice were
placed in the cylindrical glass tank (height: 80 cm, diam-
eter: 30 cm) flled with water up to a height of 40 cm [28].
The mice were not allowed to touch the bottom or jump out
of the cylinder during the test. The duration of immobil-
ity (foating with only small movements, keeping the head
above the water surface) and active swimming shown by the
mice were noted during the 6-min test session.
Statistical analysis
The data obtained were analyzed using Graph Pad Prism
using one-way or two-way ANOVA, followed by Tukey’s
post hoc test. All the results were expressed as mean SEM.
p<0.05 was considered statistically signifcant at 95% con-
fdence interval.
Open feld test (OFT) The mice were placed in the OFT
device (glass chamber consisting of 9 squares, 30 cm×30 cm
each) and observed 1 h, 3 h and 6 h post DHZ and escitalo-
pram administration for a total period of 8 min (1 min accli-
matization and 7 min testing). The arrangement of light was
set to a minimum to avoid the anxiety behaviour of the mice.
The apparatus was cleaned with 70% ethanol each time ani-
mals were removed. The mice were placed in the left corner
at the start of the trial and were observed for the number of
line crossing and centre square entry. Scoring was made for
these behavioural parameters during the entire test. OFT is
done to determine the exploratory and depressive behaviour
of the rodents [22, 29].
Results
In silico results
Prediction of drug‑likeness
Physicochemical descriptors and properties such as molecu-
lar weight, predicted central nervous system (CNS) activity,
QPlogo/w, QPlogHERG, QPlogBBB, QPlogCaco, QPlogS,
and % human oral absorption were estimated using ‘Qik-
Prop’ tool in Maestro. DHZ demonstrated a predicted per-
centage of human oral absorption of 89.89%. Predicted
apparent QPPCaco for DHZ was found to be 949.609 nm/
sec. This indicates better intestinal absorption property of
the DHZ molecule. All the pharmacokinetic properties for
DHZ showed optimum values for QPlogS, QPlogo/w and
QPlogHERG, predicting the drug-likeness of DHZ. The
results are presented in Table 1.
Tail suspension test (TST) The tail suspension test was done
as per the method suggested by Steru et al [30]. Mice were
suspended 50 cm above the foor at the edge of a table after
1 h, 3 h and 6 h of the last day of treatment with DHZ and
escitalopram. The adhesive tape was placed approximately
1 cm from the tail tip. The immobility time or absence of
escape-oriented behaviour was noted during the 6-min test-
ing period [31, 32].
Estimation of neurotransmitter
Isolation of brain and Preparation of sample After comple-
tion of the behavioural studies, mice were sacrifced, the
brain was isolated and stored at − 80 °C. Brain samples
were weighed and homogenized using ice-cold 2% for-
mic acid in water (15 ml/g), then centrifuged for 60 min at
12,000 RPM at 4 °C [33]. The supernatant was processed,
Molecular docking analysis
The molecular docking predicts binding orientation and
structural stability of the ligand–protein complex. The dock-
ing process involves the search algorithm in the generation
of diferent poses and scoring function that is involved in
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Antidepressant‑like efect of dehydrozingerone from Zingiber ofcinale by elevating…
Table 1 Predicted physicochemical properties of DHZ
Title
Mol Wt
CNS
QPlogPo/w QplogS (mol
dm–3)
QPPCaco
QPlogHERG QplogBB Rule of 5 % Human
oral absorp-
tion
Recommended
values
130.0–725.0 − 2.0 to 2.0 − 2.0 to 6.5 − 6.5 to 0.5
<25
Below − 5
− 4.288
− 3.0–1.2 Max-4
>80%: high
<25%: low
poor,>500
great
949.609
Observed values 192.214
for DHZ
0
1.650
− 2.321
− 0.688
0
89.899
pattern of the ligands with the dynamic protein in an
environment similar to physiological conditions, DHZ
was subjected to the MD simulation study for 30 ns. A
stable RMSD of 3.6 Å was observed for DHZ for 3–12 ns
duration. The formation of the H-bond and water bridge
interactions between the hydroxyl group of DHZ and resi-
due ASN181 was predominant during the 3–12 ns dura-
tion. After 12 ns, the H-bond and water bridge type of
interactions of ASN181 were lost. Stable interactions,
mainly hydrophobic and water bridge, were observed with
residue PHE208 of MAO-A and DHZ after 15 ns of the
simulation, which remained stable during 30 ns of MD
simulation. These interactions have led to a decrease in
RMSD of ligand to 1.8 Å. During the XP docking study,
there was no H-bond interaction with ILE180, ASN181,
CYS323, TYR407 and TYR444 between DHZ and MAO-
A, whereas the formation of H-bond interaction was
observed during the MD simulation study. For the entire
simulation study, residues TYR 407 (mainly H-bond and
hydrophobic interaction), TYR444 (mainly H-bond), ILE
180 (mainly H-bond and hydrophobic interaction) and ILE
335 (hydrophobic interaction) were involved in the interac-
tion with the ligand DHZ. The protein molecule was stable
throughout the simulation study. RMSD fuctuation from
1.0 to 3.5 Å was observed during the entire simulation
study. RMSF plot showing the fuctuation of 4.8 Å in the
protein structure was mainly due to the tail region residue
from 450 to 500. However, RMSF fuctuation was com-
paratively less in the case of ligand (1–2 Å), DHZ. MD
simulated interactions are represented in Fig. 2.
Fig. 1 Superposition of the inbound and redocked molecule
the evaluation of the binding interaction of diferent poses
and protein [25].
The validation of the generated grid was done by redock-
ing the inbound ligand and calculating the RMSD. RMS
of 0.5235 Ǻ was observed after the superimposition of
redocked ligand and protein inbound ligand, which vali-
dates the generated grid, as seen in Fig. 1. XP docking study
was performed which suggested that the docking score of
DHZ was better than the standard mooclobemide molecule,
as listed in Table 2.
The inhibitor binding site for MAO-A consists of the cav-
ity with a favin molecule extending from residues 210–216
[35]. The natural ligands inhibiting MAO-A has been
reported to show π–π interactions with TYR444, PHE208,
TYR407 and PHE352 as well as H-bond interactions with
ASN 181, TYR197, and TYR444 [36]. DHZ with XP score
of -7.431 showed hydrophobic interactions with TYR69,
PHE352, TYR197, TYR407, TYR444, ILE180, ILE207,
PHE208, ILE325, CYS323, ILE335, and LEU337 and polar
interactions with ASN181, GLN215, and THR336. A simi-
lar interaction was observed in moclobemide with XP score
of − 7.147 and π–π stack interaction with PHE208.
In vivo study
Characterization of dehydrozingerone
The FTIR spectra of DHZ showed absorption bands at
3311.78 cm−1 due to the OH stretching (-C6H5OH),
2999.31 cm−1 (aromatic CH stretch), 2848.86 cm−1
(aliphatic CH stretch -OCH3), 1635 carbonyl stretch-
ing (–C=C–), 1585.49 cm−1 (alkene stretch –C=C–) and
1516 cm−1 (aromatic –C=C– stretch) (Fig. 3). The ESI mass
Molecular dynamics (MD) simulation
Molecular docking can only predict the interaction of the
static protein and ligand. To understand the interaction
1 3

S. Moorkoth et al.
Table 2 Binding and
nonbonding interactions with
the XP score of DHZ and
Moclobemide
Compounds
XP Score Interactions
(kcal/mol)
2D interaction diagram
DHZ
-7.431
-
Hydrophobic:
TYR69,
PHE352,
TYR197, TYR407,
TYR444,
ILE207,
ILE325,
ILE180,
PHE208,
CYS323,
ILE335, LEU337
Polar:
ASN181, GLN215,
THR336
Moclobemide -7.147
π-π
Hydrophobic:
stack-
TYR69,
LEU97,
ILE180,
PHE208,
PHE208 ALA111,
TYR197,
VAL210, CYS323,
ILE325, ILE335,
LEU337, MET350,
PHE352,
TYR444,
Polar:
TYR407,
ASN181,
GLN215, THR336
spectra in thepositive ionization mode illustrated the base
peak at 193 m/z, corresponding to the molecular weight of
DHZ. (Fig. 4).
of DHZ dosing as compared to normal control animals.
Statistical values 1st day line crossing were: FDFn,DFd and p
value:: F4,13 =17.11 and p<0.0001. (Fig. 5).
Number of Centre square entry The number of centre
square entry in the normal control group was found to
be 1.80 0.20 on day 1 and 2.50 0.29 on day 7. Treat-
ment with escitalopram showed a signifcant increase in
the number of centre square entry on day 1 (F4,18 = 16.57,
p < 0.0001) and on day 7 (F4, 12 = 16.41, p = 0.0027) as
compared to normal control animals. DHZ treatment also
showed a signifcant increase in the number of centre
square entry on day 1 after 1 h (F4,18 = 16.57, p = 0.0005)
and 3 h (F4,18 = 16.57, p = 0.0141) dosing compared to
normal control animals. However, no signifcant change
was observed in this parameter after 6 h of DHZ treatment
(F4,18 =16.57, p=0.2098) compared to normal control ani-
mal on day 1. On day 7, DHZ treatment did not show any
signifcant change in the number of centre square entry
after 1 h (F4, 12 = 16.41, p = 0.3723), 3 h (F4, 12 = 16.41,
p = 0.3723) and 6 h (F4, 12 = 16.41, p = 0.1675) of dosing
as compared to normal control animal. Statistical values
were: FDFn,DFd and p value:: F4,18 = 16.57 and p < 0.0001
Efect of DHZ treatment in OFT
Number of Line Crossings The number of line crossings in
the normal control group was found to be 6.75 1.32 on day
1 and 4.25 0.48 on day 7. The treatment with escitalopram
showed a signifcant increase in the number of line crossings
on day 1 (F4,12 =14.37, p=0.0023) and day 7 (F4,13 =17.11,
p=0.0004) as compared to normal control. DHZ treatment
did not show any signifcant change in the number of line
crossings on day 1 after 1 h (F4,12 =14.37, p=0.9187), 3 h
(F4,12 =14.37, p=0.8140) and 6 h (F4,12 =14.37, p=0.1320)
of DHZ dosing compared to normal control. Statistical
values 1st day Line crossing was: FDFn,DFd, where DFn is
degrees of freedom numerator, and DFd is degrees of free-
dom denominator and p value:: F4,12 =14.37 and p=0.0002.
Similarly, no significant change in line crossing was
seen on day 7 after 1 h (F4,13 = 17.11, p = 0.898), 3 h
(F4,13 =17.11, p=0.8613) and 6 h (F4,13 =17.11, p=0.2929)
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Antidepressant‑like efect of dehydrozingerone from Zingiber ofcinale by elevating…
Fig. 2 Molecular dynamics simulation of MAO-A with DHZ. a 2D interaction diagram of DHZ during MD simulation. b Plot showing the inter-
action of MAO-A and DHZ molecule throughout the simulation. c RMSD plot of DHZ and MAO-A protein complex
on 1st day centre square entry, and F4, 12 = 16.41 and
p < 0.0001 for 7th day centre square entry. (Fig. 5).
FDFn,DFd and p value:: F4,24 =19.53 and p<0.0001 for FST at
day 1; F4,18 =76.82 and p<0.0001 for FST at day 7. (Fig. 6).
TST The immobility time in normal control animals
were 3.32 0.07 s and 3.33 0.05 s on 1st day and 7th
day respectively. The immobility time of mice was sig-
nifcantly reduced 1 h post DHZ treatment (F4,22 = 19.02,
p < 0.0001 and F4,17 = 18.68, p = 0023) and escitalopram
treatment (F4,22 = 19.02 with p < 0.0001 and F4,17 = 18.68
with p = 0.0055) on day 1 and day 7 respectively as com-
pared to normal control. Even 3 h post DHZ treatment
showed a signifcant decrease in immobility (F4,22 = 19.02
with p=0.0124) on 1st day as compared to normal control
animals. However, no signifcant reduction in the immobil-
ity time was observed in the mice 3 h post DHZ treatment
(3.14 0.17 s) on day 7 as compared to normal control
animals. 6 h post DHZ treatment on day 1 (F4,22 = 19.02,
p = 0.9998) and day 7 (F4,17 = 18.68, p = 0.9529) did not
show a signifcant decrease in immobility time as com-
pared to normal control. Statistical values were as follows:
Efect of DHZ treatment on immobility time of mice in FST
and TST
FST The immobility time in the normal control group
was 4.89 0.22 s and 2.92 0.52 s on 1st day and 7th day
respectively. The immobility time of mice was signifi-
cantly reduced on both 1st (F4,24 = 19.53, p = 0.0008) and
7th day (F4,18 =76.82, p=0004) after 1 h of treatment with
DHZ (100 mg/kg po) and escitalopram (F4,24 =19.53 with
p<0.0001 and 1.44 0.05 s with p=0.0002 respectively)
as compared to normal control. However, there was no
signifcant reduction in the immobility time in the mice
after 3 h (4.77 0.29 s and 2.27 0.07 s) and 6 h of DHZ
(5.00 0.27 s and 5.28 0.08 s) treatment on both day 1
and day 7 respectively. Statistical values were as follows:
1 3

S. Moorkoth et al.
Fig. 3 IR data of DHZ
FDFn,DFd and p value:: F4,22 =19.02 and p<0.0001 for TST
at day 1; F4,17 = 18.68 and p < 0.0001 for TST at day 7.
(Fig. 6.)
Estimation of neurotransmitters level in the mice brain
LC–MS method was used to measure the level of serotonin,
-noradrenaline and dopamine in the mice brain. Statistically
signifcant changes were seen for interaction (F8,75 =1027
and p < 0.0001) and neurotransmitters (F2,75 = 14,671 and
p < 0.0001) within groups (F4,75 = 3514 and p < 0.0001).
Normal control animals showed 5310.4 31.48, 817.3 6.58
and 4103.5 43.43 ng/g of tissue, respectively, for dopa-
mine, noradrenaline and serotonin levels in brain homogen-
ate. Escitalopram treatment showed a signifcant increase
in dopamine (p < 0.0001), noradrenaline (p = 0.0008) and
serotonin (p < 0.0001) levels as compared to normal con-
trol. An increase in the level of these neurotransmitters was
observed after treatment with DHZ as compared to normal
control (Fig. 8). The signifcantly highest increase was found
in the mice 1 h post-treatment for dopamine (p< 0.0001),
noradrenaline (p<0.0001) and serotonin (p<0.0001) fol-
lowed by 3 h post-treatment (p<0.0001 for all three neuro-
transmitters) and 6 h post-treatment (p<0.0001, p=0.1214
and p < 0.0001 respectively) of DHZ s compared to nor-
mal control. The level of dopamine demonstrated as four
times increase in comparison to the normal control group
while serotonin levels were increased by nearly three times.
Observed neurotransmitter levels are represented as bar dia-
grams and LC–MS chromatogram in Fig. 8
LC–MS method optimization
Optimized chromatographic conditions are as follows: Col-
umn: (HILIC Kinetix 50×2.1 mm, 2.6 µm), Mobile phase:
0.1% Formic acid in water (A) and 0.1% Formic acid in
acetonitrile (B), Gradient programme: (0.0 min %A (80);
2.5 min to 5.0 min %A (70); 9.0 min to 14.0 min %A (80),
Flow rate 0.2 mL/min, Injection volume 10 µl; Column tem-
perature: 25 °C; autosampler temperature: 6 °C. The reten-
tion time for the analytes in the extracted ion chromatogram
in the MS2 mode (Fig. 7) was found to be 5.71, 6.01, 5.51
and 5.65 min respectively for dopamine, noradrenaline, sero-
tonin and isoprenaline (internal standard) [37].
Optimized mass spectrometric conditions are as follows:
Ion source: Heated Electron Spray Ionization source (HESI),
Analyzer: Ion Trap, Operating mode: Positive polarity with
SRM (Selective Reaction Monitoring), Capillary voltage:
48.48 V, Capillary temperature: 300 °C, Source heater tem-
perature: 400 °C, Sheath gas fow rate: 50 arb, Auxiliary gas
fow rate: 12 arb, Sweep gas fow rate: 0 arb. SRM transi-
tion and collision energy (CE) were as follows: dopamine
(154.12–136.86, CE 25), noradrenaline (170.00–152.30,
CE 17), serotonin (177.10–159.93, CE 30), isoprenaline
(212.00–151.89, CE 55).
1 3

Antidepressant‑like efect of dehydrozingerone from Zingiber ofcinale by elevating…
Dehydrozingerone_200812170528
Fig. 4 Mass data of DHZ
RT: 0.00 - 3.00 SM: 13G
NL: 1.79E7
TIC F: ITMS + c APCI
0.19
100
corona Full ms
90
80
70
60
50
40
30
20
10
[110.00-1000.00] MS
Dehydrozingerone_20
0812170528
0.94
1.0
1.19 1.42
1.5
1.80 1.93 2.17 2.46
2.0 2.5
2.90
3.0
0
0.0
0.5
Time (min)
Dehydrozingerone_200812170528 #43 RT: 0.18 AV: 1 NL: 3.35E6
F: ITMS + c APCI corona Full ms [110.00-1000.00]
192.99
100
90
80
70
60
50
174.96
40
30
20
10
0
163.01
161.02
176.93
194.03
202.96
179.07
180
243.04
277.09
280
284.13
267.20
260
217.02 235.15
160
200
220
m/z
240
for their antioxidative, antityrosinase [9]; cytotoxic and anti-
microbial activity, [15, 16] Isolation of DHZ can be done
from the dried powdered rhizomes of ginger using hot meth-
anol followed by chloroform partition as suggested by Kuo
et al. [9]. This study provides insight into the involvement of
MAO-A enzyme in catalysing the deamination of the neu-
rotransmitters (chiefy dopamine, serotonin and noradrena-
line) in the antidepressant activity of DHZ, supported by
the shreds of evidence from in silico simulation, pharmaco-
logical and biochemical studies. Dysregulation of the neu-
rotransmitters like dopamine, serotonin and noradrenaline
in CNS has been proven to play an important role in the
pathogenesis of depression [6, 21, 41]. Currently, most of
Discussion
Ginger has been proven to possess antibacterial, antidiabetic,
antiobesity activity, anti-infammatory, anticancer activity,
hepatoprotective and neuroprotective activity [38]. Zinge-
rone, one of its active constituent, has been proven to possess
an anti-infammatory property by increasing the peroxisome
proliferator-activated receptor (PPARs) [39]. The constitu-
ents of ginger, namely 6-gingerol and 6-shogaol, have been
experimentally proven to modulate signalling molecules like
STAT3, MAPK, PI3K, Bcl-2, caspases and possess chemo-
therapeutic activity in both in vitro and in vivo models [40].
Dehydrozingerone and series of its analogues were evaluated
1 3

S. Moorkoth et al.
Fig. 5 Open Field Test of 1st
Day and 7th Day. The results of
open feld test (OFT) performed
on 1st and 7th day of treatment
represented as bar diagrams.
One-way ANOVA followed by
Tukey’s multiple comparison
test was used for statistical
analysis. The data are presented
as mean SEM of six animals
where ap<0.05 as compared
to normal control, bp<0.05
as compared to escitalopram,
bp<0.05 as compared to DHZ
(1 h), cp<0.05 as compared to
DHZ (3 h), bp<0.05 as com-
pared to DHZ (6 h)
Fig. 6 Forced swim test and tail
suspension test of 1st day and
7th day. The results of tail sus-
pension test (TST) and forced
swim test (FST) performed on
1st and 7th day of treatment
represented as bar diagrams.
One-way ANOVA followed by
Tukey’s multiple comparison
test was used for statistical
analysis. The data are presented
as mean SEM of six animals
where ap<0.05 as compared
to normal control, bp<0.05
as compared to escitalopram,
bp<0.05 as compared to DHZ
(1 h), cp<0.05 as compared to
DHZ (3 h), bp<0.05 as com-
pared to DHZ (6 h)
the prescribed and efcacious antidepressants are seroto-
inhibitors which act by increasing the synaptic availability
of these neurotransmitters.
nin or noradrenaline reuptake and/or monoamine oxidase
1 3

Antidepressant‑like efect of dehydrozingerone from Zingiber ofcinale by elevating…
E:\Bhim Bahadur\...\Neurotransmitter_01
Fig. 7 Extracted-ion chroma-
RT: 0.00 - 14.04 SM: 15G
togram of neurotransmitters.
NL: 1.37E4
RT: 5.71
SN: 474
Legend: MS2 extracted ion
Base Peak m/z=
chromatogram of dopamine
(5.71 min), noradrenaline
(6.01 min), serotonin (5.51 min)
and isoprenaline (Internal stand-
ard) (5.65 min)
100
80
135.86-137.86 F: ITMS + c
ESI SRM ms2
154.12@cid25.00
[134.95-138.95] MS ICIS
Neurotransmitter_01
60
40
20
NL: 2.12E1
RT: 6.01
SN: 211
Base Peak m/z=
100
80
151.30-153.30 F: ITMS + c
ESI SRM ms2
170.00@cid17.00
[152.00-154.00] MS ICIS
Neurotransmitter_01
60
40
20
NL: 6.99E3
RT: 5.51
SN: 454
Base Peak m/z=
100
80
158.93-160.93 F: ITMS + c
ESI SRM ms2
177.10@cid30.00
[157.00-161.00] MS ICIS
Neurotransmitter_01
60
40
20
NL: 4.82E1
RT: 5.65
SN: 298
Base Peak m/z=
100
80
150.89-152.89 F: ITMS + c
ESI Full ms2
212.00@cid55.00
[126.00-176.00] MS ICIS
Neurotransmitter_01
60
40
20
0
2
4
6
8
10
12
14
Time (min)
Martinez et al., have shown that the antidepressant activ-
ity of DHZ is due to the mechanism involving serotonergic
and noradrenergic systems [22]. In this study, we have shown
the involvement of the MAO enzyme system through the
computational simulation-based study as well as by estima-
tion of the brain level of neurotransmitters. We observed that
the antidepressant activity of DHZ is likely due to inhibition
of MAO enzyme that resulted in the increase in the level of
the neurotransmitters dopamine, serotonin and noradrena-
line in the brain of the mice. LC–MS based analysis of the
mice brain homogenate also has shown a 3–fourfold increase
in neurotransmitters in the DHZ treated group of mice as
compared to the normal group. This increase in the level of
neurotransmitters might be due to the inhibition of MAO-A
enzyme as predicted by the computational simulation study.
Computational simulation-based docking studies have
become a part of the drug discovery process involving hit
identifcation to lead optimization [42]. It also helps under-
stand the biological signifcance of ligand–protein interac-
tions, the enzymatic mechanism using quantum mechanics
[43]. In the present study, we have shown for the frst time,
the strong binding afnity between Dehydrozingerone and
MAO-A enzyme as well as its stability using MMGBSA
and 30 ns MD simulation study. Physicochemical property
analysis with the Qikprop tool has predicted the possibility
of DHZ to cross BBB and have good oral absorption with
1 3

S. Moorkoth et al.
Fig. 8 Neurotransmitter levels in whole brain. Legend: Two-way
ANOVA followed by Tukey’s multiple comparison test was used for
statistical analysis. The data are presented as mean SEM of six ani-
mals where ap<0.05 compared to normal control, bp<0.05 compared
c
to escitalopram, bp<0.05 compared to DHZ (1 h), p<0.05 com-
pared to DHZ (3 h), bp<0.05 compared to DHZ (6 h)
accepted Lipinski rule. This simulation-based study adds
up the evidence that the antidepressant activity of the DHZ
molecule is based on the inhibition of the MAO enzyme
[44].
efects on prolonged use and less signifcant diference is
observed between the efect of active drugs and placebo
[47]. There is a need for rapid-acting antidepressants with
fewer side efects [48]. Plant-based molecules might have
the potential to overcome these shortcomings. DHZ might
be one such potential candidate with the expected antide-
pressant activity.
FST and TST are the widely used models for the evalu-
ation of antidepressant activity [30, 45]. In the present
study, for the confrmation of the antidepressant activity of
DHZ, locomotor activity was also evaluated before FST to
avoid false-positive results. The immobility time was sig-
nifcantly reduced in mice with post 1 h DHZ (100 mg/kg
po) treatment and Escitalopram (10 mg/kg po) (selective
serotonin reuptake inhibitor) treatment on the frst day of
the test as compared to the normal mice, while 6 h DHZ
treatment showed no reduction in immobility time. On the
7th day of the test, further reduction in the immobility
time was observed in the group with post 1 h and 3 h treat-
ment of DHZ. This suggests the increased potential for the
antidepressant activity of DHZ in the chronic treatment
model. In addition, TST was also performed to ensure the
antidepressant activity. A similar pattern of the decrease
in the immobility time in TST was observed after DHZ
treatment. Open feld test was done to eliminate doubts
regarding the reduction in immobility time in FST and
TST, which might be due to the alterations of locomotor
activity by the study drug. No change in the locomotor
activity was observed by the dose of DHZ, which was used
in the study for antidepressant activity.
Conclusion
The present study showed a similar antidepressant efect
of DHZ (100 mg/kg po) to that of Escitalopram (10 mg/
kg po) after 7 days treatments in acute depression-like
studies namely FST, TST and OFT. The antidepressant
efect of DHZ was more prominent at 1 h after treatment
as compared to 3 h and 6 h of the treatment. The DHZ
treatment elevated the neurotransmitters serotonin, dopa-
mine and noradrenaline levels in the mice brain, which
was in corroboration with its observed antidepressant
activity. Computational-based virtual molecular docking
and molecular dynamic simulation studies were performed
to access the binding interaction of DHZ with MAO-A
enzyme. DHZ showed good stable interaction and binding
with the MAO-A enzyme. In silico molecular simulation
study is predicting and pointing on the MAO-A inhibitory
potential of DHZ as the molecular basis for the observed
antidepressant activity. However, further experimental
biological enzyme inhibiting potential of DHZ has to be
further evaluated for confrmation of the MAO-A inhibit-
ing potential of DHZ. In conclusion, DHZ, a half analogue
of curcumin, has been proven to possess antidepressant
Most of the current medications for depressions have
less success rate of approximately about 60% with no
response in some patients [46]. Current antidepressants
have short-term benefts and take a long period to show
effect. Most of these synthetic molecules show side
1 3

Antidepressant‑like efect of dehydrozingerone from Zingiber ofcinale by elevating…
Acad Sci. 2008;105:5739–44. https://doi.org/10.1073/pnas.07106
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Acknowledgements The authors thank Manipal—Schrödinger Cen-
tre for Molecular Simulations and Manipal College of Pharmaceutical
Sciences, Manipal Academy of Higher Education, Manipal, India for
providing facility to carry out this research work.
Author contributions Design of study: SM, NK; Neurotransmitter esti-
mation: NSP, SM, NK; Antidepressant study: NSP, NK; Molecular
docking: SM, YX, RS, KSRP, NK; Manuscript writing: SM, NSP, RS,
NK; Revision and correction of the manuscript: SM, YX, KSRP, NK.
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Funding Open access funding provided by Manipal Academy of
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Conflict of interest Authors declare no potential confict of interest.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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