Journal of Functional Foods 68 (2020) 103884

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Anxiolytic activity of Coriandrum sativum seeds aqueous extract on chronic T

restraint stressed mice and effect on brain neurotransmitters
Swati Sahoo, S. Brijesh
⁎

Sunandan Divatia School of Science, NMIMS (Deemed-to be) University, 3rd Floor, Bhaidas Sabhagriha Building, Bhaktivedanta Swami Marg, Vile Parle (W), Mumbai
400 056, India

ARTICLE INFO ABSTRACT

Keywords: Medicinal properties of Coriandrum sativum L. (coriander) are well documented. Its central nervous system re-
Anxiety lated activities are mentioned in Iranian traditional medicine. However, studies evaluating its anxiolytic prop-
Coriandrum sativum erties and the underlying mechanisms are lacking. We investigate anxiolytic activity of C. sativum L. seeds
Elevated plus maze mouse model aqueous extract (CSE) induced by chronic restraint stress and its effect on the neurotransmitter system. The
Light and dark mouse model
phytoconstituents were extracted using soxhlet apparatus and identified using LC-MS. The mice were orally
Anxiety index
Neurotransmitters
administered with the standard drug diazepam/CSE daily and exposed to restraint stress for two weeks.
Anxiolytic activity was assessed using elevated plus maze and light/dark transition test models on day 1 and 16.
On the 16th day, brain regions were quantitatively assessed for neurotransmitters. CSE treatment improved
exploratory activity in the animal models of anxiety, and restored monoamines and GABA levels to the respective
baseline levels. Moreover, CSE reduced excitotoxic levels of glutamate in the hippocampus region.

1. Introduction
Anxiety disorders, one of the most prevalent mood disorders present
themselves in the form of excessive and irrational fear, and uneasiness
to a threatening or a potentially threatening situation. Typical symp-
toms include high blood pressure, tachypnea, chest pain, heart palpi-
tations, increased muscle tension, sweating, and irritability which sig-
nificantly diminishes a person's quality of life (Thibaut, 2017). Anxiety
disorders are often found in conjunction with other psychiatric dis-
orders such as depression and substance abuse disorders. They are also
found to be comorbid with non-psychiatric disorders such as cardio-
vascular and respiratory disorders. In 2015, an estimated 3.6% (264
million) of global population were found to be living with anxiety
disorders. Anxiety disorders led to a total of 24.6 million years lost in
disability and loss of US$ 1 trillion per year in productivity in 2015,
making them the sixth major contributor to economy and non-fatal
health loss globally (World Health Organisation, 2016).
Treatment options include drug therapies and behavioral therapies.
Drug therapy commonly includes anti-anxiety drugs such as the ben-
zodiazepine (BZP) class, anti-depressants and beta blockers. Drug
therapy, however, can present a number of problems, including side
effects, poor success rates, withdrawal symptoms, development of tol-
erance, and acting on only a small fraction of the neurological me-
chanism involved in anxiety (Chouinard, 2004; Uzun et al., 2010).
Moreover, it has been observed that current drugs that target only a
specific type of receptor are insufficient to completely treat the condi-
tion especially in case of psychiatric disorders. Therefore, the need of
the hour is to develop a drug that can modulate more than one receptor
and maintain the balance in the neurotransmission. As an alternative
approach complementary and alternative medicines are gaining popu-
larity, especially in developing countries where a large population still
relies on traditional medicine due to their safer profile and are used for
the management of various psychiatric conditions including anxiety
disorders (Sahoo and Brijesh, 2019). Many modern medicines are
bioactive compounds of herbal products and their derivatives.
The Apiaceae, commonly known as parsley family, consists mostly
of aromatic plants with medicinal properties. Several plants of this fa-
mily are condiments or vegetables and have unlimited culinary appli-
cation. Also, the extracts of various plants are included commonly in
Japanese Kampo medicine. Coriandrum sativum L. (Coriander), a
member of the Apiaceae family, is a popular herb with versatile ap-
plications. It is indigenous to the Mediterranean region, and is widely
cultivated in central and eastern Europe and many Asian countries. The
leaves and seeds are widely used for culinary as well as medicinal ap-
plications. The health benefits include properties such as antibacterial,
anti-inflammatory, digestive, diuretic, carminative, anaphrodisiac, hy-
poglycemic, hypotensive, and myorelaxant etc. (Momin et al., 2012).
Some of these medicinal properties such as antibilious, tonic, diuretic,

⁎
Corresponding author.
E-mail addresses: brijeshsuku@gmail.com, brijesh.sukumaran@nmims.edu (S. Brijesh).

https://doi.org/10.1016/j.jff.2020.103884
Received 31 October 2019; Received in revised form 24 February 2020; Accepted 29 February 2020
Available online 13 March 2020
1756-4646/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
S. Sahoo and S. Brijesh
carminative and stimulant have been mentioned in Ayurveda. Anti-
anxiety and anti-epileptic activities have been indicated in the Iranian
traditional medicine (Hūšang, 2011). The sedative and hypnotic effects
have also been demonstrated scientifically (Emamghoreishi and
Heidari-Hamedani, 2015). The present study was therefore aimed to
evaluate the antianxiety activity of C. sativum and its underlying me-
chanism of action. The animal behavior was evaluated using validated
animal models such as elevated plus maze and light and dark paradigm.
Further, neurotransmitter levels were quantitatively estimated in dif-
ferent regions (hippocampus, cerebral cortex, and cerebellum and brain
stem) of the brain.
2. Materials and methods
2.1. Drug solutions and reagents
Diazepam hydrochloride was procured as a gift sample from
Centaur Pharmaceuticals Pvt. Ltd., Mumbai, India. Sodium carbox-
ymethyl cellulose was purchased from Merck specialities, Pvt. Ltd.,
Mumbai, India. γ-aminobutyric acid (GABA), serotonin (5-HT), nor-
epinephrine (NE), dopamine (DA), and Trazodone hydrochloride were
purchased from Sigma-Aldrich, Mumbai, India. DNS (1-dimethyl ami-
nonaphthalene sulfonyl chloride) was purchased from Alfa Aesar,
Mumbai, India. Glutamate, anhydrous potassium dihydrogen ortho-
phosphate (KH2PO4), Folin-Ciocalteau reagent, and ortho-phosphoric
acid (H3PO4) were purchased from Molychem, Mumbai, India, and
glacial acetic acid was purchased from SRL Diagnostics, Mumbai, India.
Other reagents were analytical grade and mobile phase chemicals were
HPLC grade. Double-distilled, deionized water was used for all studies.
Gallic acid and quercetin were purchased from LOBA Chemie and SD
Fine Chemicals, Mumbai, India, respectively.
2.2. Collection and authentication of plant material
The study was conducted using aqueous extract of C. sativum seeds,
for which the seeds were purchased from local market, Maharashtra,
India, in the month of January and authenticated at the Blatter
Herbarium, St. Xavier’s College, Mumbai, Maharashtra, India. The
voucher specimen has been deposited under the accession number
SKW. 3538.
2.3. Pharmacognostic evaluation
Physicochemical evaluation of C. sativum seed powder was carried
out, wherein loss on drying, total ash, and acid insoluble ash values
were determined based on standard analytical procedures (Mukherjee,
2008).
2.4. Preparation of extract
C. sativum seeds were subjected to size reduction using a mixer to
coarse powder. The powdered seeds were extracted using infusion
technique. Briefly, C. sativum seed powder (50 g) were soaked in water
for 24 h and the mixture was brought to boil. C. sativum seed aqueous
extract (CSE) thus obtained was filtered and concentrated by drying in
an oven at 50 °C. The dried CSE was stored in properly labeled capped
glass vials at −20 °C until used.
2.5. Preliminary qualitative phytochemical analysis
Different classes of photochemical constituents such as alkaloids,
tannins, flavonoids, saponins, anthraquinone and cardiac glycosides,
carbohydrates, and proteins were detected by subjecting CSE to pre-
liminary qualitative screening (Khandelwal and Sethi, 2014).
2
Journal of Functional Foods 68 (2020) 103884
2.6. Procurement of animals and ethics approval
Male Swiss Albino mice (weighing 20–25 g; age: 7–8 weeks) were
purchased from the Bharat Serums and Vaccines Ltd., Mumbai, India.
The animals were housed in perspex cages (6 animals/cage) and
maintained under controlled animal house conditions (25 ± 2 °C
temperature, 50%–60% relative humidity and with 12 h:12 h light and
dark illumination cycle). The animals were acclimatized to laboratory
condition for one week before the start of the experiment. The use of
laboratory animals in this study was approved (CPCSEA/IAEC/SOS/P-
05/2015) by the Institutional Animal Ethics Committee. All experi-
ments were carried out in compliance with the guidelines of the
Committee for the Purpose of Control and Supervisions of Experiments
on Animals (CPCSEA), Ministry of Environment, Forests and Climate
Change, Government of India.
2.7. Identification of phytoconstituents of CSE by LC-MS analysis
The LC-MS analysis of CSE was performed using Shimadzu 8040
(Kyoto, Japan) attached with UV detector, binary pump (G4220B), and
ESI as an ionization source. Five microliter of CSE (1 mg/mL in me-
thanol) was injected into the thermofisher C8 column
(5 µm × 150 mm × 4.6 mm) with the oven temperature set to 35 °C.
The mobile phase consisted of solvent A: aqueous 0.1% formic acid and
solvent B: acetonitrile run with a gradient program. The standardized
gradient method was programed as, time (min)/%B–0/5; 20/95; 25/95;
26/5; 30/5 with stop time at 35.0 min to ensure all compounds get
eluted out. The flow rate was set to 1 mL/min. The MS used was Triple
Quadrupole in positive and negative ion mode. The optimized MS/MS
parameters were as follows: Gas temperature: 250 °C; Gas flow: 13 mL/
min; nebulizer: 35 psi; MS range: 50–1000 m/z. LabSolutions software
was used for data acquisition. The phytoconstituents were identified by
comparing their mass and fragmentation pattern.
2.8. Behavioral tests
2.8.1. Experimental design
To investigate the anxiolytic like effects of CSE, the animals were
divided into five groups (n = 6 per group): naive (0.5% carboxymethyl
cellulose), diazepam (1 mg/kg) treated, CSE (100, 200, 400 mg/kg)
treated. Selection of dose for CSE was done on the basis of previously
reported toxicology data for aqueous extract of C. sativum seeds. CSE
has been reported safe upto 2000 mg/kg, without any signs of toxicity
and mortality (Bhat et al., 2014). Also, various pharmacological activ-
ities have been reported in the range of 50–800 mg/kg (Al-Snafi, 2016).
The drugs including diazepam and CSE were prepared freshly by sus-
pending in 0.5% CMC and were orally administered using a gavage
needle. On the first day the mice groups were dosed with the respective
drug solutions or vehicle and were subjected to behavioral paradigm
using EPM followed by LDT model, an hour after dosing. From day 2 to
day 15 (14 days) the mice in all the groups were dosed with CSE or
standard drug diazepam and exposed to restraint stress after an hour for
2 h/day (10 a.m.–12p.m.). On day 16 the mice were dosed and sub-
jected to EPM and LDT test after an hour. The mice were not exposed to
restraint stress prior to EPM and LDT on day 16. Each behavioral
evaluation was performed between 9 a.m.–12p.m. on the experimental
day. For estimation of neurotransmitter another group of untreated
mice (n = 6 per group) was used as control.
2.8.2. Elevated plus-maze test (EPM)
Behavior in the EPM is used as a means for assessing exploratory,
anxious, and motor behaviors. The EPM consists of four arms, two open
(30 × 5 cm2) and two closed (30 × 5 cm2), arranged in such a way that
the two arms of each type are opposite to each other. The maze is
elevated 30 cm above the floor. The walls of the closed arms are 12 cm
in height. The open and closed arms had a light intensity of 100 and
S. Sahoo and S. Brijesh
40 lx respectively. During the test each animal was then placed at the
center of the EPM facing one of the enclosed arms. During a 6 min test
period, the time spent in open and enclosed arms, was recorded. The
anxiety index was calculated by the equation:
1 [(open arm time/total time on the maze) + (open arm entry/total entry)]
2 was in the range 0.003–1 ppm, 0.0002–0.5 ppm, and 0.03–1 ppm, re-
The index values range from 0 to 1, with a higher value indicating
increased anxiety (Dornellas et al., 2018). Entry into an arm was de-
fined as the point when the animal placed all four paws into the arm.
After the test with each animal, the maze was carefully cleaned with
10% ethanol solution and allowed to dry before the next animal was
tested (Bourin et al., 2007).
2.8.3. Light-dark transition test (LDT)
LDT has been widely used for the evaluation of anxiety in mice
(Bourin et al., 2007), which is based on the innate aversion of rodents to
brightly illuminated areas and on the spontaneous exploratory behavior
of the animals by applying mild stressors like novel environment and
light. Therefore, LDT was used to investigate the effects of CSE on an-
xiety of mice under restraint stress. The light/dark box
(46 × 36.5 × 32 cm3) consisted of two chambers connected by an
opening (7.5 × 7.5 cm2) located at the floor level in the center of the
dividing wall. The small chamber (19 × 36.5 × 32 cm3) was painted
black and the larger chamber (27 × 36.5 × 32 cm3) was painted white.
The small chamber was covered on top to make it completely dark.
During the test, the mice were placed at the center of the light com-
partment with their back to the dark compartment, and then transition
behavior over 6 min including the total time spent visiting the light
compartment, was observed. After 6 min, mice were removed from the
box and returned to their home cage. The maze was then cleaned with a
solution of 10% ethanol and allowed to dry between tests.
2.8.4. Restraint stress protocol
The restraint stress experiments were carried out as per the method
described by Jeong et al. with minor modification (Jeong et al., 2013).
Mice were placed in 50 mL plastic falcon tubes with a small hole at the
base to provide sufficient ventilation. On the 16th day, all the mice
were subjected again to the EPM and LDT models. Non-stressed mice
were handled for 2 min daily and then returned to their home cage.
2.9. Estimation of neurotransmitters
2.9.1. Removal, isolation, and homogenization of brain regions
Following behavioral examination, mice were euthanized im-
mediately by cervical dislocation. Brain was removed quickly and
dropped in ice cold 0.1 M perchloric acid in a petri plate. The brain was
dissected and the cerebral cortex, hippocampus, and the remaining
brain tissue (cerebellum and brain stem) was individually weighed and
homogenized in 1 mL of ice cold 0.1 M perchloric acid. The resulting
mixture was centrifuged at 21,000g (Eppendorf 5810 R, Rotor F-45-30-
11) for 30 min at 4 °C. The supernatant was filtered through 0.45 μm
membrane (PTFE syringe filter, JSIL) and stored at − 80 °C until the
time of analysis.
2.9.2. Estimation of monoamines, NE, DA, and 5-HT
Monoamine levels in cerebral cortex, hippocampus, and remaining
brain tissue (cerebellum and brain stem) was estimated using HPLC
(Shimadzu, LC-2010C HT, autosampler) with fluroscence detector (RF-
20A-prominence, Shimadzu) method. Samples were injected and the
chromatographic separation was achieved with the mobile phase con-
sisting of sodium dihydrogen orthophosphate (0.01 M; pH 3.92) ad-
justed with phosphoric acid, on reversed-phase analytical column
(WATERS, C18, 5 μm, 250 mm × 0.46 mm), at room temperature. Flow
rate was set to 0.8 mL/min. NE, DA, and 5-HT were detected at an
3
Journal of Functional Foods 68 (2020) 103884
excitation wavelength of 280 nm and an emission wavelength of
315 nm. The acquired data was processed using LC Solution 151®
software. The monoamines in the samples were identified and quanti-
tated by comparing their retention time and area of the peaks with that
of the respective standard peaks. The linearity for NE, DA, and 5-HT
spectively.
2.9.3. Estimation of GABA and glutamate
Brain homogenates were thawed just before the experiment. The
reaction mixture was prepared by adding 50 µl of the sample and 20 µl
of internal standard, trazodone to 50 µl of 2 M KHCO3-KOH solution
(pH 9.8) in a centrifuge tube. 50 µl of DNS was added to the reaction
mixture. The tube was capped and the contents were mixed vigorously
for 5 s, and were reacted in the dark at 80 °C in a water bath for 30 min.
Then, 20 µl of acetic acid was added into the tube to stop the reaction
and the reaction mixture was cooled to room temperature. Five mi-
crolitre of the reaction mixture was injected directly into the HPLC
system. The mobile phase consisted of methanol and water in the ration
35:65 (v/v). The flow rate was 0.8 mL/min. The chromatographic
system consisted of a Series G1311A pump and a diode array detector
G1314A (Agilent 1100 series, Mumbai, India). The column used was
thermofisher C8, 5 µm, 150 mm × 4.6 mm (Thermofisher,
Massachusetts, United States). The wavelength used for detection of the
derivatives was 254 nm. All the measurements were performed at room
temperature. The linearity for both glutamate and GABA was in the
range of 0.05–50 ppm.
2.10. Statistical analysis
Data were presented as mean ± SD values. One-way analysis of
variance ANOVA followed by Dunnett’s post-test was performed using
Prism version 5.00 (GraphPad Software, Inc., USA). P value < 0.05
(P < 0.05) was considered as significant.
3. Results
3.1. Pharmacognostic analysis of C. Sativum seeds
Preliminary pharmacognostic analysis revealed 10.28 ± 0.002%
moisture content. Total ash and acid insoluble ash was found to be
3.7466 ± 0.008% and 0.143 ± 0.001%, respectively.
3.2. Phytochemical analysis and characterization of C. Sativum seeds
extract (CSE)
The percentage yield of the extract was found to be 4.3%.
Qualitative phytochemical analysis revealed the presence of alkaloids,
tannins, flavonoids, saponins, anthraquinone and cardiac glycosides,
carbohydrates, and proteins.
3.3. LC–MS analysis
Phytoconstituents identified by LC–MS analysis in CSE are, shown in
Table 1. Of the 10 compounds identified by LC–MS analysis, gallic acid,
ellagic acid, chlorogenic acid, tocopherol, rutin, reservatol, kaempferol,
and quercetin (Fig. 1) have been previously reported for their anti-an-
xiety activity (Bouayed et al., 2007; Takeda et al., 2003; Girish et al.,
2013; Okura et al., 2009).
3.4. Behavioral activity of CSE
3.4.1. Elevated plus maze test
On day 1, in the EPM test the animals in the vehicle control group
spent 105 ± 6.58 s (17.05% of the total time) in the open arms
S. Sahoo and S. Brijesh Journal of Functional Foods 68 (2020) 103884

Table 1
Identification of compounds in CSE by LC-MS.
Peak# Compound R.Time (min) Ion Mode Molecular ion Peak m/z Chemical Formula Area %

−
1 Kaempferol 10.837 [M−H] 286 C15H10O6 0.3
2 Rutin 14.277 [M + H]+ 611 C27H30O16 1.252
[M−H]− 609
3 Chlorogenic acid 15.239 [M−H]− 353 C16H18O9 0.621
4 Urolithin A 15.856 [M + H]+ 227 C13H8O4 2.323
[M−H]− 225
5 Tocopherol 16.966 [M + H]+ 417 C29H50O2 0.858
6 Resveratrol 17.547 [M−H]− 227 C14H12O3 0.277
7 Caffeic acid 18.543 [M−H]− 179 C9H8O4 2.869
8 Ellagic acid 20.916 [M−H]− 301 C14H6O8 2.978
9 Gallic aicd 22.571 [M−H]− 169 C7H6O5 3.8844
10 Quercetin 23.902 [M−H]− 447 C21H20O11 9.072

(Fig. 2a). The animals treated with CSE spent significantly longer time
period in the open arms at all doses. Maximum exploration was ob-
served at 400 mg/kg wherein the animals spent 134.4 ± 37.46 s
(37.3%; p < 0.001) in the open arms which was equivalent to that
observed in animals treated with standard drug diazepam (1 mg/kg)
wherein the animals spent 172.17 ± 24.14 s (47.82%; p < 0.001) in
the open arms. Also, significantly more number of entries was observed
at 400 mg/kg (10 entries; p < 0.001) compared to control group (4
entries) (Fig. 2b) Following restraint stress protocol, the animals in the
stress group spent 48 ± 21.29 s (13.3%). In contrast, animals that
were dosed daily with CSE along with stress for 14 days, showed sig-
nificant increase in the exploration time with a maximum increase
observed at 100 mg/kg wherein the animals spent 179.6 ± 31.95 s
(49.88%; p < 0.001) which was equivalent to that observed with the
standard drug diazepam wherein the animals spent 162.16 ± 39.5 s
(45.01%; p < 0.001) (Fig. 3a). However, no difference was observed in
the number of entries (Fig. 3b). CSE-treated groups showed lower va-
lues for anxiety index at all doses on day 1 with the lowest value ob-
served at 400 mg/kg (0.5; p < 0.001). After exposure to restraint
stress, CSE 100 and 200 mg/kg groups showed lower values for anxiety
index with the lowest value observed at 100 mg/kg on day 16 (0.56;
p < 0.001) compared to the vehicle control group (Figs. 2c and 3c).
3.4.2. Light-dark transition test
On day 1, in the LDT test, animals in the stress group spent
88 ± 32.38 s (26.71% of the total time) in the light area (Fig. 4.).
Treatment with CSE at 200 and 400 mg/kg significantly improved the
percentage time spent in the light area with a maximum increased
observed at 400 mg/kg wherein the animals spent 160.6 ± 26.57 s
(44.59%; p < 0.001) which was equivalent to that observed in animals
treated with standard drug diazepam wherein the animals spent
144.6 ± 10.4 s (40.16%; p < 0.05) in light area. Following restraint
stress protocol, the animals in the stress group spent 96.2 ± 28.87 s
(32.05% of the total time). Treatment with CSE (100 mg/kg) showed
significant increase in exploration of the light area wherein the animals
spent 149.6 ± 13.24 s (41.38%; p < 0.05) which was equivalent to
diazepam (1 mg/kg) wherein the animals spent 162 ± 13.38 s
(44.98%; p < 0.01) (Fig. 5).

Fig. 1. The chromatogram obtained from LC-MS analysis of CSE.

4
S. Sahoo and S. Brijesh Journal of Functional Foods 68 (2020) 103884

(a) (b) (c)

100 20
% time spent (open arms)
1.0

No. of open arm entries

***
80 15 0.8

Anxiety index
** **
60 *** * 0.6 *** ***
*** *** 10 ns ns
40 *** 0.4
5
20 0.2

0 0 0.0
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Groups Groups Groups

Fig. 2. Effect of CSE on behavior of mice on EPM model on day 1. a) Percentage time spent b) No. of open arm entries c) Mean anxiety index. (1) vehicle (0.5% CMC);
(2) Diazepam (1 mg/kg); (3) CSE (100 mg/kg); (4) CSE (200 mg/kg); (5) CSE (400 mg/kg). One-way ANOVA followed by Dunnett’s post-hoc test was applied for
statistical analysis. **P < 0.01 and ***P < 0.001, when compared to the stress group were considered to be significant.

(a) (b) (c)

100 20 1.0
% time spent (open arms)

No. of open arm entries

ns
80 0.8

Anxiety index
15 ns *
0.6
*** ***
60 *** ***
** ns ns
10
ns 0.4
40 ns
5 0.2
20

0 0 0.0
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Groups
Groups Groups

Fig. 3. Effect of CSE on behavior of mice on EPM model on day 16. (a) Percentage time spent (b) No. of open arm entries (c) Mean anxiety index. (1) vehicle (0.5%
CMC) + restraint stress; (2) Diazepam (1 mg/kg) + restraint stress; (3) CSE (100 mg/kg) + restraint stress; (4) CSE (200 mg/kg) + restraint stress; (5) CSE (400 mg/
kg) + restraint stress. One-way ANOVA followed by Dunnett’s post-hoc test was applied for statistical analysis. **P < 0.01 and ***P < 0.001, when compared to
the stress group were considered to be significant.

100 100
% time spent in light area
% time spent in light area

80 80

60 60
*** **
* ns
* *
40 40 ns
ns

20 20

0 0
1 2 3 4 5 1 2 3 4 5
Groups Groups
Fig. 4. Effect of CSE on percentage time spent in the light area of light and dark Fig. 5. Effect of CSE on percentage time spent in the light area of light and dark
model on day 1. (1) vehicle (0.5% CMC); (2) Diazepam (1 mg/kg); (3) CSE model on day 16. (1) vehicle (0.5% CMC) + restraint stress; (2) Diazepam
(100 mg/kg); (4) CSE (200 mg/kg); (5) CSE (400 mg/kg). One-way ANOVA (1 mg/kg) + restraint stress; (3) CSE (100 mg/kg) + restraint stress; (4) CSE
followed by Dunnett’s post-hoc test was applied for statistical analysis. (200 mg/kg) + restraint stress; (5) CSE (400 mg/kg) + restraint stress. One-
*P < 0.05 and ***P < 0.001 when compared to the stress group were con- way ANOVA followed by Dunnett’s post-hoc test was applied for statistical
sidered to be significant. analysis. *P < 0.05 and ***P < 0.001 when compared to the stress group
were considered to be significant.
3.5. Estimation of NE, 5–HT, and DA in mice brain
brain compared to the non-stressed animals (NE: 273.3 ± 22.59 ng/g;
3.5.1. Hippocampus DA: 7.17 ± 1.7 ng/g; 5-HT: 619.5 ± 93.72). CSE treatment showed
As shown in Fig. 6a, animals exposed to chronic restraint stress significant increase in levels of all three monoamines, NE, 5-HT, and DA
showed significant decrease in levels of NE (132.89 ± 46.48 ng/g; in hippocampus. Maximum increase in NE was observed at 200 mg/kg
p < 0.001), DA (1.961 ± 0.8 ng/g; p < 0.0001), and 5-HT (332.41 ± 33.46 ng/g; p < 0.001) and that of 5-HT at 200 mg/kg
(117.6 ± 25.89 ng/g; p < 0.0001) in the hippocampus region of (361.30 ± 83.69 ng/g; p < 0.001) and DA at 400 mg/kg

5
S. Sahoo and S. Brijesh Journal of Functional Foods 68 (2020) 103884

A
NE 5-HT DA
1000 800 15

Concentration (ng/g)

Concentration (ng/g)
Concentration (ng/g)

800 *** 600

10 *** ***
600 *** **
400 ** * **
400 *** 5
** ns 200 ### ns
200 ###
###
0 0 0
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Groups
Groups Groups

B
300 800 200
Concentration (ng/g)

Concentration (ng/g)

Concentration (ng/g)
ns
600 150 ns
200
ns
*** ** ### #
ns
ns
* 400 100

100 ***
###
ns 200 50
*** ***
0 0 0
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Groups Groups Groups

C
600 1000 50
***
Concentration (ng/g)
Concentration (ng/g)

Concentration (ng/g)
*** 800 40
400
600 30
** ###
### ***
ns 400 20
200 *** *
*
ns
200 *** 10 ###
*** ***
0 0 0
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Groups Groups Groups

Fig. 6. Effect of CSE on NE, 5-HT, and DA levels in (A) hippocampus, (B) cerebral cortex, and C) cerebellum, and brain stem. (1) vehicle (0.5% CMC); (2) vehicle
(0.5% CMC) + restraint stress; (3) diazepam (1 mg/kg) + restraint stress; (4) CSE (100 mg/kg) + restraint stress; (5) CSE (200 mg/kg) + restraint stress; (6) CSE
(400 mg/kg) + restraint stress. Values expressed as mean ± SEM in ng/g brain weight; (n = 6) in each group; One-way ANOVA followed by Dunnett’s post-hoc test
was applied for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001 when compared to normal control, and #P < 0.05, ##P < 0.01, ###P < 0.001 when
compared to untreated control were considered to be significant.

(7.23 ± 2.49 ng/g; p < 0.001). Diazepam (1 mg/kg) treatment also 32.06 ± 8.05 ng/g; 5-HT: 688.8 ± 98.4 ng/g). CSE treatment re-
showed significant increase in levels NE (644.2 ± 160.95 ng/g; sulted in a significant increase in levels of NE and DA but not in 5-HT
p < 0.001), 5-HT (289.3 ± 76.79 ng/g; p < 0.01), and DA levels. Maximum increase in levels of NE and DA was observed to be at
(7.10 ± 2.13 ng/g; p < 0.001). 400 mg/kg (NE: 471.18 ± 39.23 ng/g; p < 0.001 and DA:
19.7 ± 3.98 ng/g; p < 0.001). Diazepam treatment also showed
3.5.2. Cerebral cortex significant increase in levels of NE (358.9 ± 77.62 ng/g; p < 0.01)
As shown in Fig. 6b, animals exposed to chronic restraint stress and DA (17.84 ± 9.63 ng/g; p < 0.05) in cerebellum and brain stem
showed decrease in levels of NE (59.2 ± 13.3 ng/g; p < 0.001), DA area, however no effect was observed on levels of 5-HT.
(78.18 ± 21.68 ng/g; p < 0.001), and 5-HT (324.35 ± 46.21 ng/g;
p < 0.001) in the cortex region of the brain compared to the non- 3.6. Estimation GABA and glutamate in mice brain
stressed animals (NE: 217.1 ± 32.3 ng/g; DA: 141.3 ± 12.8 ng/g; 5-
HT: 481.7 ± 96.1 ng/g). CSE treatment resulted in increase in levels of 3.6.1. Hippocampus
compared to that of the control group. However, CSE could not recover As shown in Fig. 7a, animals exposed to chronic restraint stress
the levels of dopamine and serotonin in the cerebral cortex. Maximum showed a significant increase in glutamate levels
increase in levels of NE was observed at 200 mg/kg (NE: (324.54 ± 131.27 μg/g; p < 0.001) in hippocampus compared to the
122.37 ± 27.40 ng/g; p < 0.001). Diazepam treatment resulted in non-stressed animals (192.7 ± 21.30 μg/g). Following restraint stress
significant increase in levels of NE (109.4 ± 28.94 ng/g; p < 0.05), protocol, CSE treatment (100 mg/kg) resulted in significant reduction
but not of 5-HT and DA. (128.67 ± 55.42 μg/g; p < 0.001) in glutamate levels as was ob-
served with 1 mg/kg of diazepam (184.6 ± 47.87 μg/g; p < 0.05).
3.5.3. Cerebellum and brain stem area Chronic restraint stress reduced GABA (18.7 ± 4.21 μg/g; p < 0.001)
As shown in Fig. 6c, animals exposed to chronic restraint stress levels in the hippocampus region of the brain in animals exposed to
showed decrease in levels of NE (164.28 ± 76.28 ng/g; p < 0.001), restraint stress (Fig. 7a). CSE treatment resulted in increased GABA
DA (7.629 ± 0.781 ng/g; p < 0.001), and 5-HT (433.36 ± 48.5 ng/ levels in hippocampus in a dose dependent manner with a maximum
g; p < 0.001) in the cerebellum and brain stem region of brain com- increase (73.32 ± 21.81 μg/g; p < 0.01) observed at 400 mg/kg.
pared to the non-stressed animals (NE: 379 ± 63.8 ng/g; DA: Diazepam treatment also showed significant increase

6
S. Sahoo and S. Brijesh Journal of Functional Foods 68 (2020) 103884

A
600
Glu GABA
150
**
Concentration ( g/g)

Concentration ( g/g)
##
400 100
ns **
* ** ** **
200 50
***
##

0 0
1 2 3 4 5 6 1 2 3 4 5 6
Groups Groups
B
800 100
***
Concentration ( g/g)

Concentration ( g/g)
*** 80
600

60
400 ** **
** **
ns 40
##
200 **
20 ###

0 0
1 2 3 4 5 6 1 2 3 4 5 6
Groups Groups
C

800 150
Concentration ( g/g)
Concentration ( g/g)

**
600 ***
100 **
***
400 ** **
**
50 **
200 ## ##

0 0
1 2 3 4 5 6 1 2 3 4 5 6
Groups
Groups

Fig. 7. Effect of CSE treatment on glutamate and GABA levels in (A) hippocampus, (B) cerebral cortex, and (C) cerebellum and brain stem. (1) vehicle (0.5% CMC);
(2) vehicle (0.5% CMC) + restraint stress; (3) diazepam (1 mg/kg) + restraint stress; (4) CSE (100 mg/kg) + restraint stress; (5) CSE (200 mg/kg) + restraint stress;
(6) CSE (400 mg/kg) + restraint stress. Values expressed as mean ± SD in ng/g brain weight; (n = 6) in each group; One-way ANOVA followed by Dunnett’s post-
hoc test was applied for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001 when compared to normal control; #P < 0.05, ##P < 0.01, ###P < 0.001
compared to untreated control were considered to be significant.

(49.01 ± 12.43 μg/g; p < 0.01) in the GABA levels.
3.6.2. Cerebral cortex
As shown in Fig. 7b, animals exposed to restraint stress showed a
significant reduction in glutamate levels (160.98 ± 55.26 μg/g;
p < 0.01) compared to the non-stressed animals
(518.23 ± 270.98 μg/g). CSE treatment resulted in a significant in-
crease (299.306 ± 74.35; p < 0.01 and 577.05 ± 154.76 μg/g;
p < 0.001) in glutamate levels at 200 and 400 mg/kg respectively, in
animals exposed to chronic stress, which was similar to that observed
with 1 mg/kg of diazepam (350.20 ± 91.78 μg/g; p < 0.01). Simi-
larly, restraint stress decreased GABA levels (12.63 ± 2.35 μg/g;
p < ) in the cerebral cortex region of the brain (Fig. 7b). CSE treatment
resulted in increase in the GABA levels at all doses with a maximum
increase (71.73 ± 18.47 μg/g; p < 0.001) observed at 400 mg/kg, as
was observed when treated with 1 mg/kg of diazepam
(42.20 ± 12.01 μg/g; p < 0.01).
3.6.3. Cerebellum and brain stem
As shown in Fig. 7c, animals exposed to chronic restraint stress
showed a significant reduction in glutamate levels
(153.46 ± 20.88 μg/g; p < 0.001) in cerebellum and brain stem
compared to the non-stressed animals (339.7 ± 38.54 μg/g). CSE
treatment resulted in a significant increase in glutamate levels at all
doses with a maximum increase (455.4 ± 211.89 μg/g; p < 0.001)
observed at 100 mg/kg, as was seen with 1 mg/kg of diazepam
(429.90 ± 33.11 μg/g; p < 0.001). In addition, chronic restraint
stress significantly decreased GABA levels in the cerebellum and brain
stem region (25.75 ± 2.61 μg/g; p < 0.01) compared to the non
stressed group (56.10 ± 6.7 μg/g) as shown in Fig. 7c. CSE treatment
significantly increased GABA levels at all doses with a maximum in-
crease (94.54 ± 53.75 μg/g; p < 0.01) observed at 100 mg/kg, as
was seen with 1 mg/kg of diazepam (70.66 ± 8.40 μg/g; p < 0.01).

7
S. Sahoo and S. Brijesh
4. Discussion
In the current study, EPM and LDT animal models were employed to
analyze the effect of CSE on the anxiety. Both these animal models have
face, construct and predictive validity. This means that the clinical ef-
fectiveness to anxiolytic agents or the opposite effects to anxiogenic
agents, and the underlying cause for the response in these animal
models are similar to that observed in humans. Both these models are
known to induce anxiety in animals and are widely used for screening
anxiety modulating drugs and for investigating the psychological and
neurochemical basis of anxiety. Rodents have a natural aversion for
open spaces and EPM model uses this conflict between exploration and
aversion to open spaces. Apart from a novel environment, EPM also
provides fear of height to the rodent. Anxiolytic agents such as BZPs
(e.g., diazepam) suppress the aversive response to open spaces on EPM
model. The LDT model, on the other hand, is based on the conflict
between the tendency to explore a novel environment and aversion to
open (potentially risky) and bright spaces. Similar to EPM model,
treatment with anxiolytic drugs show increased exploration of the light
box.
Further, chronic restraint stress for two weeks was used as a model
to maintain an anxiety state in mice. Restraint stress is a type of phy-
sical stressor which is capable of activating the hypothalamic-pituitar-
yadrenal (HPA) axis of stress response. It has been previously reported
that chronic restraint stress, enhances fear memory and increases an-
xiety like behavior in the animal models such as EPM, LDT, and open
field test (Padovan and Guimaraes, 2000; Rao and Sadananda, 2016). In
this model the mice is completely immobilized daily for 2 h for a period
of 2–4 weeks.
In the present study, CSE showed significant anxiolytic activity in
mice on the EPM and LDT model on day 1 as well as on day 16 fol-
lowing repeated dosing for 14 days in the restraint stress model as was
observed with standard diazepam with significant increase in ex-
ploratory activity compared to mice in the untreated stress group. It has
been previously reported that some classes of compounds such as BZPs
and drugs acting on 5-HT1 receptors show anxiolytic effects on LDT
(Gogas et al., 2006). Therefore, CSE might be acting through the same
pathway as that of Diazepam to exhibit anxiolytic activity.
The development of anxiety disorders involves changes in various
regions of the brain, which coordinates cognitive, autonomic, motor,
and endocrine systems. The regions of the brain cerebral cortex (Wang
et al., 2016; Osuch et al., 2000), hippocampus (Rajmohan and
Mohandas, 2007; Engin and Treit, 2007; Kalisch et al., 2006; Cominski
et al., 2014) cerebellum and brain stem (Apps and Strata, 2015; Strata,
2015) were chosen for their reported involvement in anxiety and be-
havioral disorders.
Several neurotransmitter systems are affected in anxiety disorders
which mainly involve changes in their baseline levels, receptor ex-
pression and signaling, and enzymatic modulation. Clinical treatment
of anxiety disorders with pharmaceutical agents involves restoring the
function of these neurotransmitter systems, especially GABA and 5-HT.
Recently, other systems such as NE, DA, and glutamate have also been
explored. GABA has been shown to relieve stress and anxiety, and to
increase the production of alpha waves which are associated with in-
creased relaxation (Abdou et al., 2006). In contrast, glutamate, which is
an excitatory neurotransmitter, works in physiological equilibrium with
GABA, and together they are involved in 90% of neurotransmission.
Glutamate is ubiquitously found in central nervous system and is in-
volved in essential processes, including neurodevelopement and
learning, and also plays a role in acute and chronic neurodegenerative
processes. Recent findings have also revealed the underlying role of
glutamate pathway in stress response and anxiety disorders. GA-
BA–glutamate homeostasis is important for maintaining psychological
balance. An imbalance (e.g., glutamate excitotoxicity) in this can result
in various disease conditions including anxiety. In the present study, it
was observed that animals exposed to restraint stress showed decreased
8
Journal of Functional Foods 68 (2020) 103884
levels of glutamate in the cortex and brain stem whereas, increased
levels of glutamate were observed in hippocampus. This could be due to
excess secretion of glucocorticoids (GCs) under prolonged stress con-
ditions which results in hippocampal cell damage and impairment of
their ability to remove damaging excitotoxic levels of glutamate from
the synapse. Both diazepam and CSE treatment could bring the gluta-
mate levels back to normal, indicating glutamatergic pathway might be
involved in the anxiolytic action of both diazepam and CSE.
In the present study, it was observed that chronic restraint stress
exposure reduced GABA levels in cortex, hippocampus, and brain stem
region of the brain. A decrease in GABA levels in cortex, hypothalamus,
and olfactory bulb regions have been previously reported following
application of stressors such as chronic cold stress, wherein alterations
in the expression of GABA synthesizing enzymes, glutamic acid dec-
arboxylases (GAD65/67) have been suggested as the possible mechan-
isms (Acosta et al., 1993). Another study reported a decrease in GABA
levels in the cortex region due to increased GABAergic activity fol-
lowing acute and repeated immobilization/cold stress (Losada, 1988).
Studies have shown that treatment with anxiolytic botanicals such as
Ginkgo biloba; Centella asiatica and Melissa officinalis increase GABA
levels in brain (Savage et al., 2018). In the present study it was ob-
served that treatment with CSE recovered the levels of GABA in all the
regions tested as that with the standard drug diazepam, indicating that
both diazepam and CSE reduce symptoms of anxiety possibly through
GABAergic pathway.
It has been reported that different stress procedures affect the
monoaminergic transmission in various brain regions which leads to
alterations in behavior. In the present study, following chronic restraint
stress, decrease in the levels of NE, DA, and 5-HT was observed in
cortex, hippocampus, cerebellum and brain stem. Similar findings have
been reported previously, wherein decrease in the levels of 5-HT and
DA was observed in the hippocampus region following restraint stress
(1 h/day) for 40 days (Torres et al., 2002; Ahmad et al., 2010) which is
suggestive of an increase in monoaminergic activity. It has been re-
ported that hippocampus has a rich network of GCs and 5-HT receptors;
therefore increase in sertonergic activity is attributed to GC feedback
mechanisms (Chalmers et al., 1993). Kulkarni and Juvekar (Kulkarni
and Juvekar, 2008) showed similar decreases in levels of NE and DA in
the brain following application of cold restraint stress (4 °C for 1 h for
7 days) (Kulkarni and Juvekar, 2008). The levels of both NE and DA
increased significantly when treated with Nelumbo nucifera leaf aqueous
extract and standard drug diazepam. Similarly, in the present study,
CSE treatment also resulted in significant increase in DA and NE levels
in all the regions of brain tested. However, diazepam treatment resulted
in significant increase in levels of DA only in hippocampus, cerebellum
and brain stem but not in the cortex region. It has been previously re-
ported that BZPs increase the frequency and amplitude of DA release in
the nucleus accumbens area of cerebral cortex (Schelp et al., 2018).
Boireau, Dubedat, Laduron, Doble, and Blanchard (Boireau et al., 1990)
studied various compounds (with high affinity for BZP receptors) for
their effect on DA metabolism in prefrontal cortex and striatum and
found that diazepam at higher doses (10 and 40 mg/kg) increased DA
levels but not at doses below 2.5 mg/kg (Boireau et al., 1990). Hence, it
is possible that since diazepam was used at a concentration of 1 mg/kg
in the present study, it did not show any significant increase in levels of
DA in these regions. CSE treatment also showed a similar pattern for DA
which points towards a BZP like mechanism of anxiolysis. CSE treat-
ment also showed an increase in 5-HT levels in only the hippocampus
region of the brain compared to the stress exposed animals as that
observed with the standard drug diazepam. Similar results have been
previously reported with St. John’s Wort (SJW), wherein an increase of
172% in the levels of 5-HT was observed in hippocampus following
treatment with SJW in the forced swimmimg test exposure model (Ara
and Bano, 2009).
Phytochemical analysis of CSE revealed presence of various classes
of compounds such as alkaloids, tannins and phenols, flavonoids,
S. Sahoo and S. Brijesh
saponins, anthraquinone and cardiac glycosides, carbohydrates and
proteins. Phytochemical classes such as flavonoids, phenolics, alkaloids,
terpenes, and steroids have been reported to possess anxiolytic activity
(Sarris et al., 2013; Hosein Farzaei et al., 2016). Further LC-MS analysis
of CSE showed presence of compounds, which have been previously
reported for their anxiolytic activity such as chlorogenic acid (Bouayed
et al., 2007), caffeic acid (Takeda et al., 2003), ellagic acid (Girish
et al., 2013), tocopherol (Okura et al., 2009), gallic acid (Dhingra et al.,
2012; Salehi et al., 2018), quercetin (Bhutada et al., 2010; Vissiennon
et al., 2012), reservatol (Ge et al., 2016; Li et al., 2018) and rutin
(Hernandez-Leon et al., 2017). The essential oil fraction of C. sativum
seeds that contain monoterpenoids such as linalool, limonene, and
myrcene has been previously reported for sedative and motor relaxant
effects in mice (Do Vale et al., 2002). Linalool, which is the major es-
sential oil component (65%–83%) has shown anxiolytic and sedative
effect in mice and neonatal chicks (Souto-Maior et al., 2011; Harada
et al., 2018; Gastón et al., 2016). However, in the present study, since
the extract was dried in an oven at 50 °C, linalool and other volatile
components of the essential oil may have been lost. This was also
supported by the data from the LC-MS analysis, wherein presence of any
volatile oils in CSE was not detected. Hence, it may be stated that the
observed anxiolytic activity in the study could be majorly attributed to
the nonvolatile fraction of CSE. The mechanism of their anxiolytic ac-
tivity, however, still remains to be elucidated.
5. Conclusion
CSE reduces anxiety by increasing monoaminergic activity as it was
observed with the decreased levels of DA, NE and 5-HT in hippo-
campus, cerebral cortex, cerebellum and brain stem regions of brain.
Reduced GABA levels were observed in all region of the brain following
chronic restraint stress, which is indicative of progression of anxiety to
a depressed state and CSE was able to ameliorate this condition. Also,
high levels of glutamate were found in the hippocampus region in the
chronic stress group which suggests that there was neuronal damage in
the region and results with CSE suggest reversal of this damage. The
pattern of changes in neurotransmitters with CSE treatment was ob-
served to be like that of the BZP drug diazepam. Overall, the results
indicate that CSE exhibit anxiolytic properties, possibly through its
effect on GABA-glutamate and monoaminergic pathways.
6. Ethics statement
The use of laboratory animals in this study was approved (CPCSEA/
IAEC/SOS/P-05/2015) by the Institutional Animal Ethics Committee.
All experiments were carried out in compliance with the guidelines of
the Committee for the Purpose of Control and Supervisions of
Experiments on Animals (CPCSEA), Ministry of Environment, Forests
and Climate Change, Government of India.
CRediT authorship contribution statement
Swati Sahoo: Conceptualization, Methodology, Data curation,
Writing - original draft. S. Brijesh: Visualization, Investigation,
Supervision, Writing - review & editing.
Declaration of Competing Interest
None.
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