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Received: 5 May 2021    Revised: 25 June 2021    Accepted: 30 June 2021
DOI: 10.1111/jfbc.13863
ORIGINAL ARTICLE
Menthol, a bioactive constituent of Mentha, attenuates motion
sickness in mice model: Involvement of dopaminergic system
Uma Maheswari Deshetty | Anand Tamatam
Nutrition, Biochemistry & Toxicology
Division, Defence Food Research
Laboratory, Mysore, India
Correspondence
Anand Tamatam, Nutrition, Biochemistry &
Toxicology Division, Defence Food Research
Laboratory, Siddarthanagar, Mysore 570011,
India.
Email: anand@dfrl.drdo.in
J Food Biochem. 2021;45:e13863. wileyonlinelibrary.com/journal/jfbc |
https://doi.org/10.1111/jfbc.13863
 | Mahantesh Mallikarjun Patil
Abstract
Motion sickness (MS) occurs due to contradicting vestibular and visual inputs to the
brain causing nausea and vomiting. Antidopaminergic drugs being effective in re-
ducing MS create a path for effective therapy against MS by regulating dopamine
levels. We aimed to evaluate the role of the striatum and brainstem dopamine and
dopamine D2 receptor (DRD2) in MS and the efficacy of menthol (MNT) to modulate
dopamine and DRD2 in vitro and in vivo for possible amelioration of MS. Evaluation
of efficacy of MNT to inhibit dopamine release from PC12 cells and anti-­MS efficacy
in BALB/c mice model was performed. Dopamine, DRD2 expression in PC12 cells,
mice striatum, and brainstem were detected using HPLC-­ECD, RT-­PCR, and Western
blot analysis, respectively. DRD2 expression increased in calcium ionophore-­treated
PC12 cells compared with control cells. Pretreatment with 50 μg/ml menthol de-
creased dopamine and DRD2 expression. Similarly, dopamine and DRD2 levels in
mice striatum and brainstem of MS group (rotation induced) increased significantly
compared with control group NC (no rotation). Pretreatment with menthol at 50 mg/
kg concentration (rotation induced) showed decreased dopamine and DRD2 expres-
sion, thus indicating ameliorative effect on MS. Hence, we suggest that increased
striatum and brainstem dopamine and DRD2 levels might lead to MS symptoms, and
menthol could be used as a potent herbal alternative medicine for MS.
Practical applications
1. Antidopaminergic drugs being effective in reducing motion sickness (MS) creates
a path for effective therapy against MS by regulating dopamine levels.
2. Increased striatum and brainstem dopamine and Dopamine D2 receptor (DRD2)
levels might lead to the MS symptoms induced by rotation stimulation in mice
model.
3. Menthol showed a prophylactic effect on rotation-­induced MS by reducing stri-
atal and brainstem dopamine levels, DRD2 mRNA, and protein expression.
4. Menthol could be used as an herbal alternative to antidopaminergics to minimize
the associated adverse effects.
KEYWORDS
brainstem, dopamine, dopamine D2 receptor, menthol, motion sickness, striatum
© 2021 Wiley Periodicals LLC.     1 of 11
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1 |  I NTRO D U C TI O N
Motion sickness (MS) is described by a set of autonomic symptoms
which is caused by incongruent sensory inputs under conditions
of motion (Koch et al., 2018). Neural mismatch theory explains
that the intersensory contradiction between visual and vestib-
ular systems initiates the release of acetylcholine and histamine
which activates the vestibular nuclei in the brainstem leading to
nausea and vomiting (Reason, 1978; Takeda et al., 2001). This the-
ory was elucidated based on evidence that antihistaminics and
anticholinergics are the widely used medications for MS (Koch
et al., 2018).
It is reported that dopamine is the key biomarker involved in
the etiology of nausea and vomiting and subsequently dopamine
receptor D2 (DRD2) antagonists such as metoclopramide, dom-
peridone, and sulpiride are being used as antiemetics for chemo-
therapy and apomorphine-­i nduced emesis (Carpenter et al., 1984;
Leslie et al., 1990; Sakata et al., 1986). Nausea and vomiting are
the prominent symptoms of MS which paved the way to the hy-
pothesis that antidopaminergics might be effective against MS.
However, certain reports stated against the involvement of do-
pamine and DRD2 in the motion-­induced emesis and also the in-
effectiveness of metoclopramide against MS (Kohl, 1987; Takeda
et al., 1993). Contrary to this, it was reported that dopamine re-
ceptor antagonists are effective in reducing MS in animals but not
in humans (Lucot, 1998).
However, it was established that DRD2 antagonists metoclo-
pramide and sulpiride reduced MS symptoms in human patients
and squirrel monkeys, respectively (Miller & Brizzee, 1987; Rubio
et al., 2011). Moreover, metoclopramide exhibits antiemetic action
by exhibiting DRD2 antagonism at chemoreceptor trigger zone
(CTZ) in central nervous system (CNS) and is also known to show
antiemetic effect by 5-­hydroxytryptamine receptor 3 (5HT3) an-
tagonism. Since metoclopramide binds more avidly to the DRD2,
a very high dosage of metoclopramide is required to show an anti-
emetic effect through 5HT3 antagonism (Holland & Pollock, 2010).
Concurrent with this, Takeda et al. (1989) also postulated that the
two major pathways of dopaminergic nervous system, mesolimbic
and nigrostriatal pathways, might be involved in MS. Therefore, it
might be postulated that the antiemetic effect of dopamine receptor
antagonists might be by affecting the DRD2 through the nigrostria-
tal or mesolimbic pathway. Hence, the role of dopamine, DRD2 and
DRD2 antagonists in motion-­induced emesis is not clearly defined
and needs to be elucidated.
Additionally, it is also known that though potential antiemetics
DRD2 antagonists, such as metoclopramide and sulpiride, are consid-
ered to be effective against MS, these are accompanied by adverse
side effects, such as sedation, restlessness, orthostatic hypotension,
headache, confusion, focal dystonia, and tardive dyskinesia, which
limits their long-­term use among travelers (Baldessarini, 1990; Flake
et al., 2004). Hence, herbal extracts and plant-­derived bioactive
compounds have gained attention as an alternative medicine for
managing MS.
Menthol [5-­m ethyl-­2-­(1-­m ethyl ethyl) cyclohexanol; 2-­
isopropyl-­5-­m ethylcyclohexanol or p-­m ethan-­3-­ol, MNT] is a nat-
urally occurring cyclic monoterpene alcohol majorly found in the
Mentha species. Presently, MNT is widely used in the cosmetic
industry, pharmaceuticals, oral hygiene products, confectionery,
pesticides, and as a flavoring agent (Oz et al., 2017). Regarding
its medicinal uses, MNT-­
containing medications are currently
available for conditions such as gastrointestinal disorders, respi-
ratory diseases, musculoskeletal pain, and common cold (Patel
et al., 2007). MNT is known to exert potent antiemetic properties
by acting on the 5HT3 receptor by binding to a modulatory site
(Heimes et al., 2011). MNT is also known to reduce the dopamine
neuron firing frequency in the midbrain (Henderson et al., 2016).
Hence, the present study was aimed to evaluate the role of the
striatum and brainstem dopamine and DRD2 levels in MS and the
efficacy of MNT to modulate dopamine and DRD2 levels in vivo
for the possible amelioration of MS.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Chemicals and reagents
Menthol, dopamine, calcium ionphore (A23187), metoclopramide, horse
serum, fetal bovine serum (FBS), RPMI-­1640, penicillin-­streptomycin
solution, 3-­(4,5 dimethylthiazol-­2-­yl)-­2,5diphenyltetrazolium bromide
(MTT), EDTA, citric acid, triethylamine, DL-­10-­camphorsulfonic acid,
saccharin, RIPA lysis buffer, protease cocktail inhibitor, p-­coumaric
acid, and luminol were purchased from Sigma Aldrich (St. Louis, MO,
USA). Methanol, Dimethylsulfoxide (DMSO) and disodium hydrogen
orthophosphate were purchased from SRL (Mumbai, MH, India).
ELISA kits of adrenocorticotropic hormone (ACTH), cortisol, and cor-
ticosterone were procured from Enzo Life Sciences Inc. (New York,
NY, USA) and Cayman Chemical (Ann Arbor, MI, USA), respectively.
Arginine vasopressin (AVP) ELISA kit was purchased from Elabscience,
Hubei, China. Primary antibody (rabbit antidopamine receptor D2,
PAA673Mu01) and secondary antibody (HRP-­labeled Goat antirab-
bit IgG) were purchased from Cloud-­Clone Corp. (Houston, TX, USA).
SV total RNA isolation system and High-­capacity cDNA reverse tran-
scription kits were procured from Promega (Madison, WI, USA) and
Applied Biosystems (Waltham, MA, USA), respectively.
2.2 | In vitro analysis
2.2.1 | Cell maintenance and pretreatment
Rat pheochromocytoma (PC12) cells were procured from National
Centre for Cell Sciences, Pune, India. PC12 cells were grown in an
RPMI-­1640 growth medium supplemented with 10% horse serum,
5% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells
possessing 95% cell viability were utilized for experiments. Cells
DESHETTY et al.
were pretreated with different concentrations of MNT for 1 hr in
serum-­free media.
2.2.2 | Cell viability assay
Cell viability of PC12 cells was evaluated by MTT assay. Cells were
seeded in 24-­well plate at a density of 1 × 106 cells/well and grown for
24 hr. Treatment of cells with MNT (20–­140 µg/ml concentration range)
was carried out and incubated for 24  hr. After treatment, MTT was
added and assay performed according to the method described in our
previous study (Deshetty et al., 2020). The concentration of MNT pro-
viding 50% inhibition (IC50) was evaluated from the graph of concentra-
tion of MNT versus inhibition percentage in a dose-­dependent manner.
2.2.3 | Lactate dehydrogenase (LDH) leakage assay
PC12 cells were seeded, grown in 24-­well plate, and were treated
with 20 µg/ml–­140  µg/ml concentration range of MNT. After 24 hr,
LDH activity was estimated and expressed as U/L using the method
demonstrated in our prior study (Deshetty et al., 2020).
2.2.4 | Measurement of dopamine release
PC12 cells (1 × 106 cells/well) were grown in 6-­well plate for 24 hr. The
growth medium was replenished with serum-­free media after 24 hr.
Treatment of cells with different concentrations of MNT (12.5 µg/ml
[12.5 MNT], 25 µg/ml [25 MNT], and 50 µg/ml [50MNT]) was carried
out, incubated for 1 hr followed by replacing the culture medium
with stimulation buffer containing 5 µM of A23187. Cells were in-
cubated for 2 hr at 37℃ with 5% CO2 (Maheswari et al., 2020). Cell
supernatant was collected and centrifuged for 5 min at 8,000 rpm.
0.4 N perchloric acid was added to the cell supernatant and filtered
through 0.22 µm filter (Bieger et al., 2002).
Dopamine released in the cell supernatant was estimated using
HPLC (Electrochemical detector) as described in our previous study
(Maheswari et al., 2020). The concentration of dopamine in cell su-
pernatant samples was evaluated by comparing the peak area of the
samples with the peak area of standard dopamine. The concentra-
tion of dopamine was expressed as ng/ml of sample.
2.3 | In vivo study
2.3.1 | Animals
Animal study was conducted as per the guidelines of Committee
for the Purpose of the Control and Supervision of Experiments on
Animals (CPCSEA). This experiment was approved by the DFRL IAEC
(Institutional Animal Ethical Committee), approval number (IAEC-­
2016/BN/1) and comply with ARRIVE guidelines. Female BALB/c
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mice (20–­25  g) were obtained from Central Animal House Facility,
DFRL, Mysore. Mice were housed in polycarbonate cages at 25 ± 2℃,
12 hr light/dark cycle, and fed with water and pellets ad libitum.
2.3.2 | Induction of MS
Grouping
Thirty-­six BALB/c mice (female) were randomly divided into six
groups (n  = 6) such as normal control (NC, devoid rotation), MNT
at 50 mg/kg body weight control (MNT50, devoid rotation), motion
sickness (MS, induced rotation), metoclopramide (standard drug) at
20 mg/kg body weight + rotation (MCD + MS), MNT at 25 mg/kg
body weight + rotation (MNT25 + MS), and MNT at 50 mg/kg body
weight + rotation (MNT50 + MS).
Rotation device
MS was induced in mice by clockwise rotation at an angular velocity
of 50 rpm for 30 min using a custom-­designed centrifuge machine
with the specifications as described in our previous study (Deshetty
et al., 2020).
Experimental procedure
Conditioned taste aversion (CTA) method involving the usage of sac-
charin solution (SS, 0.15%) was used as an index of MS. The method
of two bottle choice test (TBCT), which includes a choice between
saccharin solution (SS) and drinking water (DW) being provided
to mice (Ossenkopp & Frisken, 1982). The experimental period of
21 days was divided into two phases (conditioning phase and test
phase) as described in our prior study (Deshetty et al., 2020).
Conditioning phase. Mice were conditioned to the water deprivation
schedule of 4 hr until the 7th day in a manner similar to our earlier
study, with the exception of rotation provided to groups MS,
MCD + MS, MNT25 + MS, and MNT50 + MS (Deshetty et al., 2020).
Test phase. In this phase, mice were provided with 4 hr water
deprivation schedule, which included oral intubation with MCD or
MNT at 3 hr of the schedule. After 4 hr, mice were provided access
to SS and DW for 1 hr. This was immediately followed by rotation
(30 min, 50 rpm) to mice from MS, MCD + MS, MNT25 + MS, and
MNT50 + MS groups. Again, mice were given access to DW/SS and
consumption was measured for the next 2 hr (Deshetty et al., 2020).
2.3.3 | Evaluation of behavioral parameters
Determination of consumption of saccharin solution/drinking water
The volume of SS/DW consumed by mice belonging to different groups
before and after rotation was calculated using the following formula:
Volume SS∕DW intake before MS induction
% SS∕DWintake = × 100
Volume SS∕DW intake after MS induction
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Behavioral scoring test
The behavioral scoring test was performed after 30 min of rotation
as per the method described by Yu et al. (2007) with minor modi-
fications. The sum of all scores assigned to each symptom was ex-
pressed as the MS index (Deshetty et al., 2020).
Open field test
Open field test (OFT) known as an animal behavioral test was per-
formed to detect the spontaneous locomotor activity of mice after
30 min of rotation (Brown et al., 1999). The specifications of the ap-
paratus used and the protocol followed are as described in our previ-
ous study (Deshetty et al., 2020).
Mice were sacrificed instantly after rotation on the 21st day of
the experimental protocol. Brain and blood samples were collected
for analysis. Mice were anesthetized and blood was collected retro-­
orbitally followed by cervical dislocation to confirm euthanasia. Brain
was harvested out and further microdissected to obtain specific stri-
atum and brainstem regions. However, the test was performed on
every alternate day in the test phase for six times which also involved
three sessions of OFT that was conducted immediately after the
rotation to avoid adaptation to MS. Therefore, a sufficient gap was
provided between the experimental trials to prevent the adaptation
process. Data were represented by calculating an average of all the
values from the experimental trials performed in the test phase.
2.3.4 | Estimation of dopamine levels
The brainstem and striatum samples were homogenized in 0.9% per-
chloric acid, centrifuged for 15 min at 10,000 rpm. Supernatants were
separated, filtered with 0.22-­μm filters, and the filtrate was analyzed
for dopamine levels using HPLC analysis (Alburges et al., 1993). The
concentration of dopamine in the samples was calculated by com-
paring the peak area of the samples with the peak area of standard
dopamine and expressed as ng/g tissue.
2.3.5 | Estimation of stress hormones
The levels of stress hormones, such as ACTH (Enzo Life Sciences Inc.,
New York, NY, USA), cortisol and corticosterone (Cayman Chemical,
MI, USA) in plasma samples, were estimated using a 96-­well plate
ELISA kit. Plasma AVP was detected using 96-­well plate ELISA kit
(Elabscience, Hubei, China). The assays were performed as per the
instructions of the manufacturer and the concentration of stress
hormones was calculated and represented as pg/ml plasma samples.
2.3.6 | Total RNA Isolation, cDNA synthesis, and RT-­
PCR analysis
PC12 cells (1 × 10 6) were seeded and grown in 25 cm2 flasks.
Cells were treated with MNT as described earlier and the cell
pellet was lysed using the RNA lysis buffer. Mice brainstem and
striatum samples were lysed using RNA lysis buffer. Total RNA
was extracted and converted to cDNA as described in our pre-
vious study using the specific kits. Housekeeping gene used
for comparison was β-­a ctin. Primers targeting the DRD2 gene
in both rat PC12 cells and mice striatum and brainstem sam-
ples were KiCqStart primers purchased from Sigma Aldrich (St.
Louis, MO, USA). The primer sequences used for RT-­P CR were
rat DRD2: sense primer 5′-­C AACAATACAGACCAGAATGAG-­3′,
antisense primer 5′-­G GAGGACGATGTAGATTTTG-­3′; mouse
DRD2: sense primer 5′-­T TGTTCTTGGTGTGTTCATC-­3′, anti-
sense primer 5′-­TATAGATGATGGGGTTCACG-­3′; rat β-­a ctin:
sense primer 5′-­A AGACCTCTATGCCAACAC-­3′, antisense
primer 5′-­TGATCTTCATGGTGCTAGG-­3′; and mouse β-­a ctin:
sense primer 5′-­G TCAAGATCATTGCTCCTC-­3′, antisense primer
5′-­T TGTCAAGAAAGGGTGTAAC-­3′. The PCR reaction mix and
reaction cycles were set up as mentioned in our previous study
using DRD2 primers. Changes in gene expression were expressed
ΔΔCt
as mean fold change by using the 2-­ method (Deshetty
et al., 2020).
2.3.7 | Western blot analysis of Dopamine D2
receptor (DRD2) protein
PC12 cells (1 × 106) were seeded 25 cm2 flasks and treated with MNT
as described earlier. The cell pellet, brainstem, and striatum samples
were homogenized in protease cocktail inhibitor-­containing RIPA
lysis buffer followed by centrifugation (10,000× g, 4℃) for 20 min.
The protein in cells and tissue homogenates was determined (Lowry
et al., 1951). Protein sample (50 µg), primary antibodies (GAPDH and
DRD2), and secondary antibody (HRP-­labeled Goat antirabbit IgG)
were used to perform the western blotting analysis, and band in-
tensity was measured as mentioned in our previous study (Deshetty
et al., 2020).
2.4 | Statistical analysis
The in vitro data were represented as mean ±  SEM of re-
sults calculated from three independent experiments con-
ducted in triplicates, respectively. The in vivo data were
represented as the mean ±  SD of results attained from three
experimental trials (n  =  6), respectively. GraphPad Prism ver-
sion 6 software (LaJolla, CA, USA) was used for the statistical
analysis. The MS group was compared with NC group (control)
and MCD + MS, MNT25 + MS, MNT50 + MS groups were
compared with the MS group. Data were analyzed by one-­
way ANOVA and comparisons among different groups were
made using Tukey's multiple comparisons test, where *p  < .05,
**p  < .01, ***p  < .001 compared with NC group and #p  < .05,
##
p  < .01, ###
p  < .001 compared with MS group was considered
as significant.
DESHETTY et al.
3 | R E S U LT S
3.1 | In vitro analysis
3.1.1 | Effect of MNT on PC12 cell viability
The IC50 value of MNT in PC12 cells was 100 µg/ml (Figure 1a).
Based on the IC50 value, the concentration of MNT was further fixed
for dopamine release assay.
3.1.2 | Effect of MNT on LDH leakage
Control PC12 cells released 30.85 ± 4.11 U/L LDH and MNT treated
PC12 cells released between 56.99 ± 8.11 and 289.46 ± 11.18 U/L.
The LDH release increased with increase in the MNT concentration
(Figure 1b). This in turn indicates the cytotoxicity of MNT at differ-
ent concentrations and IC50 value.
3.1.3 | Effect of MNT on dopamine release from
PC12 cells
Control PC12 cells released 47.28 ± 4.99 ng/ml, whereas the cal-
cium ionophore (A23187)-­treated cells released 377.43 ± 10.16 ng/
ml. Cells pretreated with 50 MNT exhibited inhibition in dopamine
release to maximum extent (57.12 ± 9.12 ng/ml) compared with
12.5 MNT (165.78 ± 11.11 ng/ml) and 25 MNT (64.50 ± 8.12 ng/
ml) (Figure 1c).
3.1.4 | Effect of MNT on DRD2 mRNA and protein
expression in PC12 cells
PC12 cells treated with A23187 exhibited upregulated DRD2
mRNA expression by 2.074  ± 0.049 folds in comparison with the
PC12 control cells. PC12 cells pretreated with MNT showed re-
duced DRD2 mRNA expression by 1.30  ± 0.061 fold (12.5 MNT),
F I G U R E 1   Dose-­dependent effect of menthol pretreatment on (a) PC12 cell viability, (b) extracellular LDH leakage from PC12 cells (U/L),
(c) concentration of dopamine release from PC12 cells (ng/ml). Values are expressed as mean ± SEM of three independent experiments.
***p < .001 compared with PC12 control cells; ##p < .01 and ###p < .001 compared with A23187 treated cells
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1.103  ± 0.069 fold (25 MNT), and 0.71 ± 0.041 fold (50 MNT) in
comparison with A23187-­treated PC12 cells (Figure  2a). Increased
DRD2 protein expression was detected in A23187-­treated PC12
cells (215.34% ± 9.33%) compared with control PC12 cells (100%).
PC12 cells pretreated with MNT showed significantly reduced the
DRD2 protein expression in 12.5 MNT (182.62% ± 6.17%), 25 MNT
(169.26% ± 7.23%), 50 MNT (124.87% ± 8.11%) compared with PC12
cells treated with A23187 (Figure 2b,c).
3.2 | In vivo analysis
3.2.1 | Effect of MNT on drinking water (DW)/
saccharin solution (SS) consumption
The SS consumption of mice from MS group (16.54% ± 0.95%) de-
creased compared with the control groups NC (26.24% ± 0.58%)
and MNT50 (25.69% ± 0.47%). Pretreatment with MCD + MS
(25.11% ± 0.68%), MNT25 + MS (20.32% ± 0.93%), and MNT50 + MS
(24.92%  ± 0.65%) increased the SS in comparison with MS group
(Figure 3a).
3.2.2 | Effect of MNT on behavioral scores
The behavioral score exhibited by mice from the MS group
(16.60 ± 1.12) was higher than the control groups NC (2.2 ± 0.68)
and MNT50 (2.20 ± 0.62). The behavioral score of mice from the pre-
treated groups MCD + MS (4.8 ± 0.66), MNT25 + MS (8.34 ± 0.72),
and MNT50 + MS (5.20 ± 0.56) reduced in comparison with MS
group (Figure 3b).
3.2.3 | Effect of MNT on open field test scores
Mice belonging to the MS group exhibited significantly increased
freezing duration and decreased rearing, number of line cross-
ings, and total distance traveled compared with the control groups
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F I G U R E 2   Effect of menthol on (a) DRD2 mRNA expression in PC12 cells, (b,c) DRD2 protein expression in PC12 cells. Values are
expressed as mean ± SEM for three trials. **p < .01 compared with control PC12 cells; #p < .05, ##p < .01, and ###p < .001 compared with
A23187-­treated PC12 cells

F I G U R E 3   (a) Volume (%) of saccharin solution (SS) and drinking water (DW) consumed per mice after rotation of 30 min; (b) behavioral
score exhibited by mice after rotation. Values are expressed as mean ± SD for six mice per group. *p < .05 and **p < .01 compared with NC
group; #p < .05, ##p < .01, and ###p < .001 compared with MS group

(NC and MNT50). Mice from the pretreatment groups MCD + MS,
MNT25 + MS, MNT50 + MS showed decreased freezing ­duration and
significantly increased rearing, number of line crossings, and total
distance traveled. The behavioral pattern was similar to the control
groups, indicating the amelioration of MS (Figure 4).
3.2.4 | Effect of MNT on dopamine levels
3.2.5 | Effect of MNT on stress hormone levels
AVP, ACTH, cortisol, and corticosterone levels in plasma samples
were increased in mice from the MS group compared with NC and
MNT50 (control groups). Pretreatment with MCD and MNT reduced
the levels in the plasma samples and were comparable with control
groups (Table 1).

Dopamine levels were increased in the striatum sample of MS group
(158.62 ± 8.68) compared with the control groups NC (72.15 ± 7.99)
and MNT50 (70.55 ± 8.56). Pretreatment with MCD + MS
(73.68  ± 7.92), MNT25 + MS (111.73 ± 7.02), and MNT50 + MS
(76.11 ± 7.54) exhibited decreased dopamine levels compared with
the MS group. Brainstem dopamine levels in NC, MNT50, and MS
groups were 50.22 ± 8.89, 48.22 ± 9.18, and 139.69 ± 9.68, respec-
tively. Pretreatment with MCD + MS (59.11 ± 7.45), MNT25 + MS
(102.46 ± 6.99), and MNT50 + MS (73.11 ± 7.54) reduced the dopa-
mine levels (Figure 5a).
3.2.6 | Effect of MNT on DRD2 mRNA expression
DRD2 mRNA expression in striatum samples of MS group was up-
regulated by 2.040 ± 0.058 folds compared with control group.
Expression was downregulated in MCD + MS (0.51 ± 0.091),
MNT25 + MS (1.11 ± 0.08), MNT50 + MS (0.75 ± 0.032) compared
with the MS group. The brainstem DRD2 mRNA expression got
upregulated in MS group by 3.11 ± 0.092 folds compared with the
control group. Whereas, in pretreatment groups, expression was de-
creased by MCD + MS (0.73 ± 0.083), MNT25 + MS (1.54 ± 0.095),
DESHETTY et al. |
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F I G U R E 4   Open field test scores exhibited by mice from different groups after 30 min rotation. (a) Number of line crossings, (b) rearing,
(c) total distance traveled (cm), and (d) freezing duration (s). Values are expressed as mean ± SD for six mice per group. *p < .05, **p < .01, and
***p < .001 compared with NC group; #p < .05, ##p < .01, and ###p < .001 compared with MS group

F I G U R E 5   Effect of MNT on (a) dopamine levels (ng/g tissue) and (b) DRD2 mRNA expression in striatum and brainstem samples of mice.
Values are expressed as mean ± SD for six mice per group. **p < .01 and ***p < .001 compared with NC group; ##p < .01 and ###p < .001
compared with MS group

MNT50  + MS (0.83 ± 0.05) folds compared with the MS group
(Figure 5b).
3.2.7 | Effect of MNT on DRD2 protein expression
Increased striatum DRD2 protein expression (185.12% ± 6.45%) was
observed in the MS group compared with the control group (100%). The
pretreated groups exhibited decreased DRD2 expression, MCD + MS
(130.46% ± 8.32%), MNT25 + MS (161.55% ± 7.11%), MNT50 + MS
(135.34%  ± 7.56%), compared with the MS group. The brainstem
DRD2 protein expression was increased to 196.28% ± 8.75% in the
MS group compared with the control group (100%). The pretreated
groups exhibited decreased DRD2 protein expression, MCD + MS
(135.62% ± 9.44%), MNT25 + MS (165.12% ± 9.46%), MNT50 + MS
(143.06% ± 8.09%), compared with the MS group (Figure 6).
8 of 11       | DESHETTY et al.

TA B L E 1   Stress hormone levels in

Corticosterone
plasma samples of mice (pg/ml sample)
Groups AVP (pg/ml) ACTH (pg/ml) Cortisol (pg/ml) (pg/ml)

NC 23.29 ± 3.86 101.61 ± 6.42 168.63 ± 9.51 96.10 ± 8.63

MNT50 22.88 ± 3.72 102.67 ± 4.66 172.43 ± 5.44 100.02 ± 7.51
MS 81.63 ± 6.43* 198.86 ± 8.11* 384.94 ± 5.98** 208.44 ± 6.56*
MCD + MS 35.67 ± 5.11###  121.22 ± 4.98##  210.34 ± 5.66 ##  120.38 ± 5.11## 
# ## #
MNT25 + MS 52.58 ± 4.89   153.23 ± 7.11   248.34 ± 6.12   143.67 ± 5.12# 
MNT50 + MS 46.34 ± 5.11##  136.37 ± 4.18##  218.27 ± 3.84 ##  128.41 ± 4.48## 

Note:: Values are expressed as mean ± SD for six mice per group.
Abbreviations: ACTH, adrenocorticotropic hormone; AVP, arginine vasopressin.
*p < .05,; **p < .01 compared with NC group;
#
p < .05,
##
p < .01,
###
p < .001 compared with MS group.

F I G U R E 6   Effect of MNT on DRD2 protein expression in striatum and brainstem samples of mice. Values are expressed as mean ± SD for
six mice per group. **p < .01 compared with NC group; #p < .05 compared with MS group

4 |  D I S CU S S I O N role of dopamine, DRD2, DRD2 antagonists in MS is obscure and

Antihistaminics and anticholinergics are the widely used class of
drugs for MS as histamine and acetylcholine elevations are consid-
ered to be the primary reason for MS as described by sensory conflict
theory (Koch et al., 2018). Dopamine has been considered to play a
role in etiology of nausea and vomiting induced by chemotherapy,
postoperative, drug (apomorphine) as DRD2 antagonists are effec-
tive by acting on DRD2 at chemoreceptor trigger zone (CTZ) in area
prostema (Holland & Pollock, 2010; Smith et al., 2012). However, the
needs to be explored. Few reports suggested the noninvolvement
of dopamine and DRD2 in motion-­induced nausea and vomiting due
to the ineffectiveness of DRD2 antagonists such as metoclopramide
(Kohl, 1987; Takeda et al., 1993). However, contrary to these antido-
paminergics such as DRD2 antagonists (metoclopramide, sulpiride)
are reported to reduce MS symptoms in animals and humans (Miller
& Brizzee, 1987; Rubio et al., 2011). Concomitant with this, Takeda
et al. (1989) hypothesized that the mesolimbic or nigrostriatal dopa-
minergic pathway might be involved in MS. In view of this, dopamine
DESHETTY et al.
might be considered as an important biomarker and its role in MS
needs to be elucidated by exploring the role of dopamine, DRD2,
and DRD2 antagonist via the nigrostriatal or mesolimbic pathway.
However, it is known that though DRD2 antagonists are potential
antiemetic and considered effective against MS, it causes side ef-
fects, such as sedation, restlessness, orthostatic hypotension,
headache, confusion, tardive dyskinesia, and focal dystonia, which
limits their long-­term use among travelers (Baldessarini, 1990; Flake
et al., 2004). Hence, natural bioactive molecules from plant origin
are gaining importance to be used as alternative medicine as they
are free from adverse side effects (Hwang et al., 2019). MNT, a cy-
clic monoterpene found majorly in Mentha species is known to have
a potent antiemetic property (Heimes et al., 2011; Thawkar, 2016).
Hence, the present study was performed to evaluate the role of the
striatum and brainstem dopamine and DRD2 in MS and the efficacy
of MNT to modulate dopamine and DRD2 levels in vitro and in vivo
for possible amelioration of MS.
PC12 cells can synthesize, store, metabolize, and release dopa-
mine in a method similar to that observed in vivo (Greene & Rein,
1977). A23187 increases the calcium ions permeability into the cells
which are essential for the release of dopamine from cells (Garcia
et al., 1975). Hence, this could be used as an efficient in vitro model
for evaluating the effect of MNT by checking the extent of inhibi-
tion on dopamine release and DRD2 expression from PC12 cells
(Maheswari et al., 2020). It was found that dopamine levels in-
creased and DRD2 expression was upregulated in cells treated with
A23187 compared with the control cells. The pretreatment with
50MNT (50 μg/ml) decreased both dopamine and DRD2 expres-
sion to a maximum extent compared with cells treated with A23187
(Figures 1 and 2). Therefore, it was established in vitro that MNT
could decrease dopamine levels and reduce DRD2 (mRNA and pro-
tein) expression.
Concomitantly, BALB/c mice model was used to evaluate the
effect of MCD and MNT on striatal and brainstem dopamine and
DRD2 levels in MS. CTA and behavioral parameters such as be-
havioral scoring and OFT were used as MS index. In the present
study, when provided a choice between DW and SS, mice from all
the groups preferred SS rather than DW before rotation. It was ob-
served that after 30 min of rotation, mice from MS group (rotation
induced) and preferred DW avoided SS compared with NC group
(control group, no rotation), thus demonstrating aversive behav-
ior toward SS. This was in agreement with prior published results
(Chen et al., 2018; Ossenkopp & Frisken, 1982). Whereas pretreated
groups MCD + MS, MNT25 + MS, MNT50 + MS preferred SS rather
than DW. Hence, indicating a protective effect of MCD and MNT at
10 and 50 mg/kg bodyweight concentration, respectively, on rota-
tion induced MS symptoms in mice (Figure 3a).
Simultaneously in behavioral scoring tests, specific symptoms
such as piloerection, urination, tremor, and defecation were ob-
served and indexed with a scoring system (Yu et al., 2007). It was
found that mice belonging to the MS group exhibited a higher
score compared with the control groups NC and MNT50. This
indicates that rotation increased piloerection, urination, tremor,
|
      9 of 11
and defecation in MS group which resembles earlier published
reports (Qi et al., 2019; Wang et al., 2017; Zheng et al., 2014;
Zhou et al., 2017). Pretreatment with MCD and MNT exhibited
a reduction in the score of the symptoms (Figure 3b). Rotation
significantly decreased the rearing, total distance traveled, num-
ber of line crossings and increased the freezing duration in the
MS group compared with the control groups (NC and MNT50),
which is in accord with a prior report (Wang et al., 2012). Whereas
mice belonging to treatment groups (MCD + MS, MNT25 + MS,
MNT50  + MS) exhibited restored spontaneous locomotor activ-
ity, determining the protective effect of MCD and MNT at 10 and
50 mg/kg b.wt. concentration, respectively, on rotation induced
MS symptoms (Figure 4).
Concurrent with this, it was found that dopamine and DRD2
mRNA and protein expression in striatum and brainstem regions
of the mice from MS group (rotation induced) upregulated signifi-
cantly compared with the NC group (control group and no rotation)
(Figures 5 and 6). This outcome may be correlated to the fact that
histamine release during MS leads to vasopressin release which in
turn induces stress (Horii et al., 2001; Kjaer et al., 1998; Sharman &
Low, 2008). It was found that levels of hormones AVP, ACTH, corti-
sol, and corticosterone increased in plasma samples of the MS group
compared with the control groups NC and MNT50 (Table 1). This may
be correlated to the fact that AVP is an etiological hormone and con-
tributes to MS development (Xu et al., 2015). AVP, in turn, activates
the hypothalamic-­pituitary-­adrenal (HPA) axis causing the release of
stress hormones (ACTH, cortisol, and corticosterone) and glucocor-
ticoids which eventually induces stress and promotes the release of
catecholamines such as dopamine (Antoni,  1993; Chrousos,  2009;
Smith & Vale, 2006). It is also reported earlier that induction of stress
causes the release of dopamine in the striatum, nucleus accumbens,
and medial frontal cortex (Abercrombie et  al.,  1989). Hence, the
stress induced due to rotation might be the reason for the release
of dopamine from the striatum which activated the DRD2 receptors
in the striatum. It is also known that dopaminergic innervations from
striatum extend to the substantia nigra compacta which is present
in the midbrain (a part of the brainstem) (Pliszka, 2016). These in-
nervations might have activated the dopaminergic neurons in the
brainstem causing the dopamine release and activation of DRD2
receptors in the brainstem which in turn activates the vomiting cen-
ter in the brainstem through DRD2 and causing symptoms of MS.
However, mice from treatment groups MCD + MS, MNT25 + MS,
MNT50  + MS (rotation induced) exhibited decreased stress hor-
mones, dopamine levels, DRD2 mRNA/protein expression compared
with the MS group (Table 1, Figures 5 and 6).
Moreover, it has been reported that elevated striatal dopamine
levels further facilitate conditioned avoidance behavior (Cooper
et al., 1974). In correlation, we found that mice from the MS group
(rotation induced) showed conditioned aversive behavior toward SS
(Figure 3a), which could be due to the increased dopamine levels in
the striatum of these mice (Figure 5a). Further, stress-­induced during
rotation stimulation might also be a possible reason for decreased
locomotor activity in the mice belonging to the MS group in the OFT
 |
10 of 11       DESHETTY et al.
(Figure 4), as stress is thought to impair vestibular compensation by
causing behavioral changes (Yamamoto, 2000). But pretreatment of
mice with MCD (10 mg/kg b.wt) and MNT (50 mg/kg b.wt) showed
the restorative effect on behavioral patterns indicating possible pro-
tective effect against MS.
Hence, the present study suggests that increased striatum and
brainstem dopamine and DRD2 levels might lead to the symptoms of
MS induced by rotation stimulation in the BALB/c mice model. We
also established that MNT showed a protective effect on rotation-­
induced MS by decreasing the striatal and brainstem dopamine and
DRD2 mRNA, protein levels in a manner almost similar to metoclopr-
amide. Thus, MNT with no known adverse effects or toxicity at this
dosage could be used as an herbal antidote and alternative medicine
to antidopaminergic medications to minimize the related adverse ef-
fects and discomfort of travelers. However, the role of dopamine
and DRD2 could also be analyzed real-­time by microdialysis as a part
of future studies. Also, the effect of MNT on other pathways such
as serotonergic and GABAergic in MS could be explored. However,
further studies in human volunteers are required to determine the
efficacy of MNT to reduce MS.
AC K N OW L E D G M E N T S
The authors extend their deep sense of gratitude to Director,
Defence Research and Development Organisation-­Defence Food
Research Laboratory (DRDO-­DFRL) for his constant encouragement
and support throughout the study.
C O N FL I C T O F I N T E R E S T
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
AU T H O R C O N T R I B U T I O N S
Uma Maheswari Deshetty: Conceptualization; Data curation; Formal
analysis; Investigation; Methodology; Writing-­original draft. Anand
Tamatam: Project administration; Supervision; Writing-­review & ed-
iting. M M Patil: Software; Validation.
DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from
the corresponding author upon reasonable request.
ORCID
Anand Tamatam  https://orcid.org/0000-0003-2387-730X
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How to cite this article: Deshetty, U. M., Tamatam, A., &
Patil, M. M. (2021). Menthol, a bioactive constituent of
Mentha, attenuates motion sickness in mice model:
Involvement of dopaminergic system. Journal of Food
Biochemistry, 45, e13863. https://doi.org/10.1111/jfbc.13863