Biomedicine & Pharmacotherapy 109 (2019) 1698–1708

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Biomedicine & Pharmacotherapy

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

Protective effect of Convolvulus pluricaulis against neuroinflammation T

associated depressive behavior induced by chronic unpredictable mild stress
in rat
⁎,1
Girdhari Lal Gupta , Joneth Fernandes1
Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM's NMIMS, V.L. Mehta Road, Vile Parle (W), Mumbai, 400 056, India

ARTICLE INFO ABSTRACT

Keywords: Depression is a heterogeneous disorder and has been regarded as an inflammatory disease. The aerial parts of the
Chronic unpredictable mild stress Convolvulus pluricaulis are used in Indian traditional medicines for the management of nervous disorders.
Convolvulus pluricaulis However, the influence of methanolic extract of aerial parts of Convolvulus pluricaulis (CPE) on a chronic animal
Depression model of depression has not been investigated yet, and associated biochemical changes are still unclear.
Cytokines
Therefore, this study investigates the effects of CPE on a chronic rat model of depression and explores its un-
Interleukin-1β
Interleukin-6
derlying mechanism of action on neuroinflammation and brain monoamines. The antidepressant-like effect of
Tumor necrosis factor-α CPE (25, 50, and 100 mg/kg, p.o.) was depicted using the sucrose preference test and the forced swimming test
ALT (FST) while CUMS-induced alteration in the locomotor index was measured using the open field test (OFT) and
AST actophotometer. A consecutive one-week treatment of CPE (50, and 100 mg/kg) or fluoxetine (10 mg/kg, p.o.)
ALP treatment significantly increased sucrose preference index, reduced immobility time in the FST, and increased
the number of squares crossed, the number of rearing in the OFT and locomotion in the actophotometer in the
CUMS-exposed rats. Moreover, elevated levels of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and liver bio-
markers ALT, AST were also significantly reversed by CPE (50, and 100 mg/kg) or fluoxetine administration in
the CUMS-exposed rats. Furthermore, a one-week treatment of CPE (50 and 100 mg/kg) or fluoxetine also re-
markably restored the serotonin and noradrenaline levels in the hippocampus as well as in the prefrontal cortex
of the CUMS-exposed rats. However, CPE (25 mg/kg) exerted insignificant protection against CUMS-induced
depressive-like behavior and associated neuroinflammation. Therefore, this study demonstrates that CPE exerted
antidepressant-like effect which could be mediated by anti-inflammatory potential, restoring liver biomarkers or
monoaminergic responses in the stressed rats.

1. Introduction depression, most of the current antidepressants used in the clinical

As stated by the World Health Organization, depression affects an
estimate of 300 million people of all ages globally and is a highly de-
bilitating, life-threatening psychiatric illness [1,2]. Depression is re-
garded to become the second most disabling burden in the World by
2030 [3,4]. Depression leads to pathological stages like anhedonia,
helplessness feelings, reduced physical activity, negative moods, weight
loss, low morale, and cognitive dysfunctions, etc. [5–7]. Despite en-
ormous progress have been made to understand the pathogenesis of
practice not only have a low efficacy, but they also may cause side
effects [8–10]. Therefore, the development of safer and effective newer
medications is always desired world-wide. Chiefly, antidepressants
based on the monoaminergic deficiency hypothesis have a key role in
the clinical practice but are also associated to depict treatment-resistant
in one-third of the patients [11–13]. However, the current focus is also
on treatment strategies that influence the distinct neurochemical sys-
tems [14]. Increasing evidence has claimed that depression is an oxi-
dative and inflammatory disorder [15–19]. Clinical evidence also

Abbreviations: CUMS, chronic unpredictable mild stress; ELISA, enzyme-linked immunosorbent assay; SPT, sucrose preference test; OFT, open field test; FST, forced
swimming test; HPTLC, high-performance thin layer chromatography; IL-1β, interleukin-1beta; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; ALT, Alanine
transaminase; AST, Aspartate transaminase; ALP, Alkaline phosphatase
⁎
Corresponding author at: Department of Pharmacology, School of Pharmacy & Technology Management, SVKM’s NMIMS, V. L. Mehta Road, Vile Parle (W),
Mumbai, 400 056, India.
E-mail address: girdhari_gupta@rediffmail.com (G.L. Gupta).
1
Both authors contributed equally to this work.

https://doi.org/10.1016/j.biopha.2018.11.046
Received 20 October 2018; Received in revised form 3 November 2018; Accepted 10 November 2018
0753-3322/ © 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
G.L. Gupta, J. Fernandes
closely associates inflammation with the complicated pathogenesis of
neuropsychiatric disorders, which include depression [4,20]. Several
reports have revealed the close association of the expression of pro-
inflammatory mediators and depression-like behavior [16,20,21].
Moreover, pro-inflammatory cytokines including interleukin-1β (IL-1
β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) have been
engrossed to increase during depression in animals [4,17,19,21]. De-
pressive patients have also depicted monoaminergic deficiency and
elevated levels of pro-inflammatory cytokines in the peripheral circu-
lation and some brain regions [20,22,23]. Although the pathogenesis of
depression progression has been elucidated extensively, further studies
are still needed to assimilate the relevant molecular mechanism. Ad-
ditionally, most of the reports on depression primarily focus on changes
in the brain areas including the hippocampus and prefrontal cortex
[24–26].
Herbal medicines are gaining attention for the development of new
therapeutically active compounds with higher potency and lower
toxicity. A perennial herb Convolvulus pluricaulis Choisy
(Convolvulaceae) commonly known as Shankpushpi has been reported
to depict multiple pharmacological activities, including improvement of
learning and memory in young and old mice [27] and clinically in
improving memory, especially in long-term memory loss in the younger
age group [28]. Convolvulus pluricaulis has ameliorated human micro-
tubule-associated protein tau-induced neurotoxicity in Alzheimer's
disease in Drosophila model [29], declined tau and amyloid precursor
protein expression [30], prevented impairment of cholinergic and an-
tioxidant activity induced by aluminium in rat brain cerebral cortex
[31], depicted neuroprotective activity [32,33], attenuated scopola-
mine-induced amnesia [34], exhibited neuroprotective activity against
tumor necrosis factor-alpha (TNF-α) and prostaglandin E2 [33], and
reduced 3- nitropropionic acid-induced neurotoxicity in rats [35]. Ad-
ditionally, Convolvulus pluricaulis also attenuated alcohol addiction in
mice [36], elicited a significant antidepressant-like effect [37], and
decreased Triton WR-1339-induced hyperlipidemia [38]. Convolvulus
pluricaulis is also known for demonstrating antioxidant, anticonvulsant
activity [39,40], anxiolytic effects [41], and antiulcerogenic effect [42].
Moreover, the plant is mentioned as one of the best Medhya Rasayana
(nervine tonic) in Ayurveda, and the aerial parts of the plant are used
locally in India for hundreds of years for various nervous disorders. This
herb is also marketed as a single or multi-herb formulation. The active
constituents of the herb include alkaloid (shankhapushpine), flavonoids
(kaempferol derivatives), phytosterol (β-sitosterol), 20-oxodo-
triacontanol, tetra-triacontanoic acid, 29-oxodotriacontanol and sco-
poletin [33,35,43,44]. Interestingly, scopoletin has been reported to
depict antidepressant-like effect through monoaminergic systems [45]
and also suppresses pro-inflammatory cytokines [46].
Chronic unpredictable mild stress (CUMS) consists of mild stressors
which imitate the common stressors in routine human life. It results in
alterations in neural, endocrine variables and behavioral changes [47].
Moreover, the influence of Convolvulus pluricaulis on a chronic animal
model of depression has not been elucidated yet, and associated bio-
chemical changes, an inflammatory link is still unclear. Therefore, the
present investigation was undertaken to confirm the antidepressant-like
effects of the methanolic extract of the aerial parts of Convolvulus
pluricaulis extract (CPE) in the CUMS animal model and to investigate
whether the underlying mechanism accountable for the beneficial ef-
fects of CPE are associated with alterations in pro-inflammatory bio-
markers in plasma and monoamines in the hippocampus and prefrontal
cortex regions of the brain.
2. Materials and methods
2.1. Drugs doses and routes of administration
Convolvulus pluricaulis (Konark Herbals, Mumbai), serotonin (Tokyo
Chemical Industry Co., Ltd., Japan), scopoletin (Sisco Research
1699
Biomedicine & Pharmacotherapy 109 (2019) 1698–1708
Laboratories Pvt. Ltd., Mumbai, India), norepinephrine (NE) (Sigma-
Aldrich, St. Louis, MO, United States), and fluoxetine (Sun Pharma,
India) were procured from the respective sources. Alanine transaminase
(ALT) kit, aspartate transaminase (AST) kit, and alkaline phosphatase
(ALP) kit were purchased from ERBA Diagnostics, Mannheim. HPTLC
grade methanol was purchased from S.D. Fine Chemical Ltd. (Mumbai,
India). All other chemicals and reagents were of analytical grades and
were purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai,
India, and Merk India Ltd., Mumbai, India. Commercial kits of Rat IL-1β
ELISA, Rat IL-6 ELISA, and Rat TNFα ELISA were purchased from
Krishgen Biosystems, India. Convolvulus pluricaulis and fluoxetine were
dissolved in 0.5% w/v of sodium–carboxymethylcellulose (Na-CMC) in
distilled water. Oral administration of CPE and fluoxetine were selected
and considered as the most preferred route of administration in the
psychiatric patients.
2.2. Animals and housing
Male Wistar rats (7–8 weeks, weighing 120–150 g) were supplied by
Bharat Serum and Vaccines Ltd, Thane, India, and housed in a con-
trolled environment with 25 ± 2 °C temperature, 70 ± 10% relative
humidity and 12 h light/12 h dark cycle in well-ventilated poly-
propylene cages. Standard commercial rodent pellets (Nutrimix
Laboratory Animal Feed, Maharashtra, India) and water were provided
ad libitum. The rats were acclimatized to the laboratory conditions for 7
days before the experiments. An approval prior to animal experi-
mentation was obtained from the Institutional Animal Ethics
Committee, constituted as per Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA), Govt. of India
(CPCSEA/IAEC/P-93/2017).
2.3. Plant material and preparation of the methanolic extract
Dried aerial parts of Convolvulus pluricaulis was purchased from
Konark Herbals, Mumbai, India. The authentication of the plant
Convolvulus pluricaulis was carried out by scientists of
Agharkar Research Institute, grant-in-aid research institute of the
Department of Science and Technology (DST), Government of India,
Pune, India. The voucher specimen (no. 1402/2018) was kept for the
future reference. The plant was coarsely powdered with a mechanical
grinder and passed through sieve number 60 and stored in a sealed
container. The coarsely powdered plant material was defatted with n-
hexane. The defatted, air-dried plant powder was further extracted in
soxhlet apparatus with methanol at 50 °C for 48 h. The use of metha-
nolic solvent has been reported to be rich in alkaloids and coumarins
[38,48]. The solvent was recovered using rotary evaporator (Heidolph,
Germany) under reduced pressure of 600 mmHg at 40 °C. The semisolid
mass was further lyophilized for 24 h. The methanolic extraction of dry
aerial plant material (1 kg) yielded 81 g (8.1% w/w) of the extract. The
lyophilized dry powder was sealed in amber bottles and stored in a 4 °C
freezer before use.
2.4. Phytochemical analysis
Preliminary phytochemical screening of CPE was carried out qua-
litatively for the presence of carbohydrates, proteins, amino acids, al-
kaloids, glycosides, flavonoids, saponins, and tannins by utilizing
standard procedures [48,49].
2.5. Quantification of scopoletin in CPE by High-performance thin layer
chromatography (HPTLC) method
The reference compound scopoletin was quantitated in CPE as re-
ported in the monograph of Convolvulus pluricaulis in Quality Standards
of Indian Medicinal Plants published by Indian Council of Medicinal
Research, Government of India, New Delhi, India [49] with slight
G.L. Gupta, J. Fernandes
Fig. 1. High-performance thin layer chromatography fingerprints of (A) stan-
dard scopoletin (Rf 0.67) and (B,C,D) scopoletin in the methanolic CPE in tri-
plicate using pre-coated silica gel aluminum plate 60 F254, mobile phase ethyl
acetate: toluene: acetic acid, 5: 4: 1, v/v/v at 300 nm.
modification using HPTLC (GmbH/SARSTEDT; Desaga, Germany), in-
cluding AS30 HPTLC applicator, CD60 HPTLC densitometer with Pro
Quant Windows software. Briefly, CPE was dissolved in methanol at a
concentration of 20 mg/ml and scopoletin at 2 mg/mL. The calibration
curve for scopoletin was prepared on a pre-coated silica gel aluminum
plate 60 F254 of 0.2 mm thickness (E. Merck, Germany). For the esti-
mation of scopoletin in the extract, one band of the standard scopoletin
solution of volume 20 μl and CPE test solution of volume 20 μl was
applied in triplicate on a precoated TLC plate. Glass twin-trough
chamber saturated with the mobile phase was used for the development
of TLC plate to a height of 80 mm. The mobile phase was comprised of
ethyl acetate: toluene: acetic acid (5: 4: 1 v/v/v). The plates were air
dried and scanned at 300 nm. The peak area under the curve was re-
corded, and a calibration curve for scopoletin was plotted. The amount
of scopoletin present in the samples was calculated from the calibration
curve. Photo documentation of scopoletin is presented in Fig. 1.
2.6. Acute toxicity study
An acute toxicity study was conducted as per the guidelines of the
Organization for Economic Co-Operation and Development 423 [50].
Female Wistar rats were fasted overnight and administered a single
dose of 0.5% Na-CMC (control group) (n = 3) or 2000 mg/kg of CPE
dissolved in 0.5% Na-CMC (n = 3) orally. Thereafter, the rats were
monitored continuously for 14 days. On the last 14 day, the rats were
Table 1
Timeline of the experimental design.
Day (-7 to 0) Day 1 - Day Day 21 - Day 27
20
Adaptation to Lab CUMS exposure (Except normal control group)
(Not counted in the Drug Treatment (10 ml/kg, p.o.
study) administration, once daily)
Non-CUMS Normal control group: vehicle
CUMS CUMS group: vehicle
CPE (25 mg/kg)
CPE (50 mg/kg)
CPE (100 mg/kg)
Fluoxetine (10 mg/kg)
CUMS, chronic unpredictable mild stress; CPE, methanolic extract of areal part of Convolvulus pluricaulis; OFT, open field test; SPT, sucrose preference test; FST,
forced swimming test; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; ALT, Alanine transaminase; AST, Aspartate transaminase; ALP,
Alkaline phosphatase.
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Biomedicine & Pharmacotherapy 109 (2019) 1698–1708
euthanized and their vital organs were individually depicted for mac-
roscopic evaluation.
2.7. Experimental design
All behavioral studies were executed between 09:00 and 15:00 h in
the naive experimental rats. After acclimatization to the laboratory
conditions, 36 rats were randomly divided into six groups: (1) Non-
stressed + vehicle (control); (2) CUMS + vehicle; (3–5) three CPE-
treated groups (25, 50, and 100 mg/kg of CPE + CUMS); and (6)
CUMS + 10 mg/kg, p.o. of standard drug fluoxetine. The doses of CPE
and fluoxetine were selected based on the previous pharmacological
reports [35,37,51–53]. CPE and fluoxetine were administrated once a
day for the last seven days of the CUMS exposure (from day 21 to day
27). The rats in the normal control group and CUMS group received an
equivalent volume of 0.5% Na-CMC (10 ml/kg, p.o., once a day, from
day 21 to day 27). One-hour after the drug administration, behavioral
tests were executed, and subsequently animals were sacrificed for fur-
ther biochemical determinations. The detailed schedule of the experi-
mental illustration is presented in Table 1.
2.8. Chronic unpredictable mild stress procedure
Rats were exposed to a variety of mild environmental or psycho-
social stressors as described previously [54,55] with minor modifica-
tions as per the schedule in Table 2. Briefly, the control group rats were
housed with 3 rats per cage with free access to food, and water and
were left unstressed in their home cages with the exception of general
cleaning and handling. However, rats in the CUMS and drug-treated
groups were isolated in the individual cages and exposed to the fol-
lowing nine stressors for 27 days: (1) Warm swimming at 40 °C for
5 min; (2) 24 h of water deprivation; (3) 24 h of the cage tilting (30°);
(4) tail pinching for 1 min (1 cm from the tip of the tail); (5) 24 h of food
deprivation; (6) overnight illumination; (7) overhang (10 min); (8) cold
swimming at 8 °C for 5 min; (9) physical restraint for 2 h. These stres-
sors were executed once daily between 09:00 h and 13:00 h (except the
stressors of 24 h) with a variable unpredictable sequence for con-
secutive 27 days and each stressor was repeated three times within 27
days. Prophesy and adjustment to any particular stressor were arrested
by not repeating the same stressor for the two consecutive days.
2.9. Behavioral tests
After the termination of the CUMS procedure, trails were executed
in a sound-attenuated room using the standard behavior paradigm in
the order listed in Table 1, i.e., open field test (OFT) on day 28, acto-
photometer test on day 29, sucrose preference test (SPT) on day 30 and
31, and forced swim test (FST) on day 33 and 34. Only one test was
Day 28 - Day 34 Day 35
Behavioral tests Sacrifice and sample collection
OFT (on day 28) Estimation of serotonin and noradrenaline levels in the
hippocampus and prefrontal cortex
Actophotometer (on day 29)
Estimation of pro-inflammatory cytokines (IL-1β, IL-6,
SPT (on day 30 and 31) and TNF-α) and
liver biomarkers (ALT, AST, and ALP) in plasma
FST (on day 33 and 34)
G.L. Gupta, J. Fernandes
Table 2
The stressors of chronic unpredictable mild stress procedure with specified
days.
Stressors Days of CUMS exposure
Warm swimming at 40 °C for 5 min 1 16
24 h of water deprivation 2 15
24 h of cage tilting (30°) 3 12
Tail pinching for 1 min (1 cm from the tip of the 4 10
tail)
24 h of food deprivation 5 11
Overnight illumination 6 14
Overhang (10 min) 7 13
Cold swimming at 8 °C for 5 min 8 17
Physical restraint for 2 h 9 18
performed each day keeping a 24 h lapse between the sucrose pre-
ference test and the forced swim test. The behavioral trials were per-
formed by a trained observer who was unaware of the experimental
groups. The use of fluoxetine as a positive control was exerted because
of its routine use in the treatment of CUMS-induced depression-like
behavior [56].
2.9.1. Open-field test
The OFT was executed under bright ambient room light to assess the
locomotor and exploratory behavior of the rats as previously reported
[55]. The rats were placed individually into the center area and allowed
to explore the unfamiliar arena for 5 min. The number of total squares
crossed, number of rearing (number of times the rat raised on its hind
legs), and grooming episodes (washing of the coat) were registered.
Post-exposure of each rat, the OFT apparatus was wiped with a 50%
ethanol to abolish any sign of olfactory cues.
2.9.2. Spontaneous locomotor activity
Locomotion of each rat was investigated in the actophotometer
(Dolphin) for the period of 5 min [57].
2.9.3. Sucrose preference test
The SPT was used to observe anhedonic-like behavior and executed
as reported earlier with minor modifications [47,58]. Initially, rats
were trained to consume 2% w/v sucrose solution for 24-hours by
placing two bottles of sucrose solution in each cage to prevent neo-
phobia-induced changes in the preference. After a 12-hour period of
food and water deprivation, all rats were given free access to two
standard drinking bottles, one containing 2.5% sucrose and the other
with tap water for the period of three hours. The sucrose and water
intake were measured, and sucrose preference is presented as the per-
centage of total volume of fluid consumed.
Volume of sucrose solution ingested
Sucrose preference % = x 100
Volume of total liquid ingested
2.9.4. Forced swim test
To ascertain the helplessness characteristic of the depression, fre-
quently used FST model was standardized in our laboratory [59–61].
Rats were subjected singly in a transparent cylinder and the immobility
time was recorded for the period of 5 min.
2.10. Blood sampling and tissue extraction
After the behavior tests on day 35, rats were anesthetized with
pentobarbital sodium (45 mg/kg) by intraperitoneal injection, and
blood was collected from the retro-orbital sinus. Ethylene-diamine-
tetraacetate coated vials were used to collect the blood sample for es-
timation of pro-inflammatory cytokine IL-1β, IL-6, and TNFα. The
plasma pro-inflammatory cytokine levels were detected in duplicate
with commercial ELISA kits according to the manufacturer's
Biomedicine & Pharmacotherapy 109 (2019) 1698–1708
instructions. The mean absorbance was determined for each set of du-
plicate standards and samples. The levels of cytokines IL-1β, IL-6, and
TNFα were expressed as pg/mL. For biochemical estimation, blood was
collected without using an anticoagulant and allowed to clot for 30 min
at 25 °C. Thereafter, centrifuged at 1500×g for 15 min at 4 °C to collect
25 the serum, which was stored at −20 °C. The level of liver enzymes ALT,
21
AST and ALP were measured using commercially available standard
24
23 biochemical assay kit. Post-blood withdrawal on day 35, rats were sa-
crificed by decapitation to remove the brain and isolated brain tissues
22 i.e. the hippocampus and prefrontal cortex. The brain tissues were
20 frozen (−80 °C) for further analysis of serotonin and noradrenaline
19
26
levels in the prefrontal cortex and the hippocampus.
27
2.11. HPLC analysis of brain tissue homogenate to measure serotonin levels
The prefrontal cortex and hippocampus were homogenized in 2 ml
of 0.1 N perchloric acid and centrifuged at 8000×g for 20 min at 4 °C
separately. The supernatant of the tissue homogenate was collected and
stored at −80 °C until use. This supernatant was further filtered using a
0.45 μm membrane filter (Durapore, Millipore) and tested for serotonin
concentration by using the HPLC method [62]. Chromatography was
performed with HPLC (SHIMADZU: LC-2010 CHT, Shimadzu, Kyoto,
Japan), equipped with 25 cm length, 4.6 mm internal diameter Kro-
masil C18 Column (particle size 5 mm) (Kromasil CA). The clear tissue
homogenate was injected in triplicates and chromatographic separation
was achieved at 280 nm using 0.05% formic acid and acetonitrile
(90:10 v/v) as the mobile phase with 1 ml/min flow rate. Standard
serotonin weighing 10 mg was diluted to 10 ml with the mobile phase
to make a 1000 μg/ml stock solution. The stock solution was further
diluted with the mobile phase to get 100, 200, 300 and 400 μg/ml so-
lutions. These four dilutions were scanned in triplicates at 280 nm, and
the chromatographs were recorded to obtain the retention time and
peak area under the curve. The calibration curve was obtained by
plotting the peak area under the curve versus concentration. Ad-
ditionally, CPE and fluoxetine-treated rat brain tissue homogenate
samples were scanned in triplicates at 280 nm. Finally, the concentra-
tion of serotonin was calculated using the calibration curve.
2.12. Statistical analysis
The data were statistically analyzed using GraphPad Prism Software
version 7.00, San Diego, California, USA, and expressed as a mean ±
standard error of the mean (S.E.M.) (n = 6). Data were analyzed by
two-way analysis of variance (ANOVA), followed by post-hoc Tukey’s
multiple comparison tests. Differences with p < 0.05 were considered
statistically significant.
3. Results
3.1. Phytochemical analysis of CPE
Preliminary phytochemical screening of CPE depicted the presence
of carbohydrates, proteins, amino acids, alkaloids, glycosides, flavo-
noids and, saponins. The HPTLC analysis depicted well-resolved peaks
of CPE revealing the presence of scopoletin. The calibration curve for
scopoletin was linear in the range of 1.0–5.0 μg/mL. The linear re-
gression equation for the calibration curve was y = 57.087x+699.34;
(R2 = 0.9907) where y is the peak area response at 300 nm and x re-
present the concentration in μg/mL. The spots of the entire chromato-
gram were visualized under 300 nm and the percentage of scopoletin
(Rf 0.67) in CPE was found to be 0.106% w/w (Fig. 1).
3.2. Acute oral toxicity studies
No mortality or morbidity was depicted in the rats during the ob-
servation period (14 days) following a single administration of CPE
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G.L. Gupta, J. Fernandes
(2000 mg/kg, p.o.). Morphological features (eyes, nose, fur, and skin)
appeared normal. No tremors, diarrhea, convulsions, salivation, le-
thargy or unusual behavior like walking backward, self-mutilation were
monitored. There were no macroscopic alterations in the brain, liver,
heart, kidney, and stomach (data presented in a supplementary sheet as
Fig. 1S). This indicates that CPE was safe up to a single dose of
2000 mg/kg, p.o. in experimental rats, and the extract was classified as
safe (category 5), according to the OECD 423 guidelines.
3.3. Effects of CUMS and CPE in the open field test
As illustrated in Fig. 2, reduction in the number of squares crossed
and the number of rearing were noticed significantly in the CUMS-ex-
posed rats, and those were reversed by one-week treatment with CPE
(50, and 100 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o.) in the CUMS-
exposed rats. Two-way ANOVA followed by post-hoc Tukey’s multiple
comparison tests depicted significant differences in the number of
square crossing, and the number of rearing [F (5, 30) = 35.06, p <
0.0001] in the OFT. Rats in the CUMS group demonstrated a significant
decline in the number of squares crossed (p < 0.0001; Fig. 2) and the
number of rearing (p < 0.01; Fig. 2) in the OFT as compared to the
normal control rats. Treatment with CPE (100 mg/kg, p.o.) or fluox-
etine (10 mg/kg, p.o.) for seven consecutive days significantly in-
creased the number of squares crossed (p < 0.001, p < 0.0001; Fig. 2),
and the number of rearing (p < 0.05, p < 0.05; Fig. 2) respectively in
the OFT as compared to the CUMS-exposed rats. CPE (50 mg/kg, p.o.)
also exerted significant reversal in the number of squares crossed (p <
0.01; Fig. 2) in the OFT as compared to the CUMS-exposed rats. How-
ever, no significant effects on the number of squares crossed, and the
number of rearing were observed on the treatment with a lower dose of
CPE (25 mg/kg, p.o.) when compared to the vehicle-treated CUMS-ex-
posed rats (p > 0.05; Fig. 2). Furthermore, there were no significant
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Biomedicine & Pharmacotherapy 109 (2019) 1698–1708
Fig. 2. Effects of CUMS and CPE treatment on
the number of total squares crossed, number of
rearing, and number of grooming episodes in
the OFT. Results are shown as mean ± SEM
(n = 6) by vertical bars, where **p < 0.01,
****
p < 0.0001 (Compared with normal con-
trol group), #p < 0.05, ##p < 0.01,
###
p < 0.001, ####p < 0.0001 (Compared
with vehicle-treated CUMS-exposed rats), Two-
way analysis of variance (ANOVA), followed by
post-hoc Tukey’s multiple comparison test.
CUMS, chronic unpredictable mild stress; CPE,
methanolic extract of areal part of Convolvulus
pluricaulis; OFT, open field test.
Fig. 3. Effect of CUMS and CPE treatment on
the locomotor index in the actophotometer.
Results are shown as mean ± SEM (n = 6) by
vertical bars, where ****p < 0.0001
(Compared to normal control group), #p <
0.05, ###p < 0.001, ###p < 0.001
(Compared to vehicle-treated CUMS-exposed
rats), Two-way analysis of variance (ANOVA),
followed by post-hoc Tukey’s multiple com-
parison test. CUMS, chronic unpredictable mild
stress; CPE, methanolic extract of areal part of
Convolvulus pluricaulis.
differences in the numbers of grooming among all CPE and fluoxetine-
treated groups (p > 0.05; Fig. 2) in the CUMS as well as normal control
rats. Additionally, the separate experiment was executed to confirm the
drugs escalating locomotor index which may endanger false-positive
behavioral results. In that study, rats were unexposed to the CUMS
procedure (normal rats), and one-week CPE administration (25, 50, and
100 mg/kg, p.o.) did not alter the total number of squares crossed (p >
0.05), the number of rearing (p > 0.05) and number of grooming (p >
0.05) in the OFT and locomotor score in the actophotometer (p > 0.05)
when compared with vehicle-treated normal control group (Data pre-
sented as supporting information, Fig. 2S & 3S). Therefore, this clearly
indicates that elevated locomotion in the OFT and reduced immobility
in the FST on the CPE treatment in the CUMS-exposed rats cannot be
regarded as a psychostimulant action.
3.4. Effect of CPE treatment in the CUMS rats on locomotion activity
As shown in Fig. 3, the influences of CUMS and CPE on the loco-
motion were also investigated using actophotometer. Application of
two-way ANOVA depicted a significant difference in the locomotion
count [F (5, 25) = 18.58, p < 0.0001]. Post-hoc Tukey’s multiple
comparison tests depicted that CUMS group showed a significant re-
duction in the locomotion count as compared to the normal control rats
in the actophotometer (p < 0.0001; Fig. 3). CPE (50, and 100 mg/kg,
p.o.) or fluoxetine (10 mg/kg, p.o.) treatment for seven consecutive
days significantly increased the locomotion count (p < 0.05, p <
0.001, p < 0.001; Fig. 3) in the actophotometer as compared to the
CUMS-exposed rats. However, no significant reversal in the locomotor
count was exerted after treatment with CPE (25 mg/kg, p.o.) as com-
pared with CUMS-exposed rats (p > 0.05; Fig. 3).
G.L. Gupta, J. Fernandes
3.5. Influences of CPE on the percent of sucrose consumption
As presented in Fig. 4, significant differences were depicted among
the treatments groups on the percent of sucrose consumption [F (5,
25) = 12.23, p < 0.0001]. Two-way ANOVA followed by post-hoc
Tukey’s multiple comparison tests demonstrated the significant de-
crease in the percent of sucrose consumption in the CUMS-exposed rats
in comparison with normal control group (p < 0.001; Fig. 4). In this
study, one-week administration of CPE (50, and 100 mg/kg, p.o.) and
fluoxetine (10 mg/kg, p.o.) revealed an antidepressant-like effect, as
percentage sucrose intake in CUMS-exposed rats significantly raised
(p < 0.05, p < 0.01, p < 0.001; Fig. 4) respectively as compared with
vehicle-treated CUMS-exposed rats. However, no significant alteration
was observed in the percentage sucrose consumption on the treatment
with CPE (25 mg/kg, p.o.) as compared with the vehicle-treated CUMS-
exposed rats (p > 0.05; Fig. 4).
3.6. Influences of CPE on depressive-like behavior in rats
In the FST (Fig. 5), two-way ANOVA revealed significant differences
among various groups in the immobility time [F (5, 25) = 49.52, p <
0.0001]. Post-hoc Tukey’s multiple comparison tests demonstrated that
CUMS-exposed rats depicted the significant increase in the immobility
time as compared to the normal control rats (p < 0.0001; Fig. 5).
Chronic treatment with the CPE (50, and 100 mg/kg, p.o.) and fluox-
etine (10 mg/kg, p.o.) significantly decreased the immobility duration
in the CUMS-exposed rats when compared with the vehicle-treated
CUMS-exposed rats (p < 0.05, p < 0.0001, p < 0.0001; Fig. 5) re-
spectively in the FST rat model. Moreover, CPE (25 mg/kg, p.o.) did not
exert any significant differences in the duration of immobility in the
CUMS-exposed rats (p > 0.05; Fig. 5).
3.7. Influences of CPE on the pro-inflammatory cytokines
Next, the effect of CPE on the pro-inflammatory cytokine levels in
the plasma were examined. Two-way ANOVA exerted significant
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Biomedicine & Pharmacotherapy 109 (2019) 1698–1708
Fig. 4. Influences of CUMS and CPE on the
percent of sucrose preference. After the ad-
ministration of CPE (25, 50, and 100 mg/kg,
p.o.), fluoxetine (10 mg/kg, p.o.) or vehicle,
rats were subjected to the SPT. Results are
shown as mean ± SEM (n = 6) by vertical
bars, where ***p < 0.001 (Compared to
normal control group), #p < 0.05, ##p <
0.01, ###p < 0.001 (Compared to vehicle-
treated CUMS-exposed rats), Two-way analysis
of variance (ANOVA), followed by post-hoc
Tukey’s multiple comparison test. CUMS,
chronic unpredictable mild stress; CPE,
methanolic extract of areal part of Convolvulus
pluricaulis; SPT, sucrose preference test.
differences in the pro-inflammatory cytokine IL-1β [F (5, 25) = 13.86,
p < 0.0001], IL-6 [F (5, 25) = 7.023, p < 0.0001], and TNFα [F (5,
25) = 12.53, p < 0.0001] in the plasma of rats as compared to the
normal control group. The post-hoc Tukey’s multiple comparison test
demonstrated that exposure of the rats to the chronic CUMS depicted
significant elevation in the pro-inflammatory cytokine IL-1β (p <
0.0001; Fig. 6A), IL-6 (p < 0.001; Fig. 6B), and TNFα (p < 0.0001;
Fig. 6C) levels in the plasma. One-week treatment with CPE (50, and
100 mg/kg, p.o.) and fluoxetine (10 mg/kg, p.o.) markedly attenuated
IL-1β (p < 0.05, p < 0.0001, p < 0.001; Fig. 6A), IL-6 (p < 0.05, p <
0.01, p < 0.05; Fig. 6B), and TNFα (p < 0.01, p < 0.0001, p < 0.01;
Fig. 6C) levels in the plasma respectively as compared to the vehicle-
treated CUMS-exposed rats. Moreover, CPE (25 mg/kg, p.o.) failed to
produce any significant effects on the pro-inflammatory cytokine levels
in the CUMS-exposed rats (p > 0.05; Fig. 6).
3.8. Influences of CPE on the liver biomarkers
The effect of CPE on the liver biomarkers were examined and pre-
sented in the Fig. 7. Two-way ANOVA revealed significant differences
in the liver biomarkers ALT [F (5, 25) = 6.11, p = 0.0008], AST [F (5,
25) = 11.71, p < 0.0001] and insignificant changes in the ALP levels
[F (5, 25) = 1.47, p=0.2350] in the plasma of rats as compared to the
normal control group. One-week CPE (100 mg/kg, p.o.) or fluoxetine
(10 mg/kg, p.o.) administration significantly decreased the elevated
level of liver enzymes ALT (p < 0.01, p < 0.05; Fig. 7A) and AST (p <
0.01, p < 0.05; Fig. 7B) levels respectively in the plasma as compared
to the vehicle-treated CUMS-exposed rats. Moreover, CPE (50 mg/kg)
showed significant changes on the ALT levels (p < 0.05; Fig. 7A) while
AST and ALP were not changed significantly in the CUMS-exposed rats
(p > 0.05; Fig. 7B & 7C). However, CPE (25 mg/kg, p.o.) did not de-
monstrate any significant changes on the ALT, AST, and ALP levels in
the CUMS-exposed rats (p > 0.05; Fig. 7). ALP levels were not changed
significantly in the all animal group (p > 0.05; Fig. 7C).
Fig. 5. Influences of CUMS and CPE on the
immobility time in the FST. After the adminis-
tration of CPE (25, 50, and 100 mg/kg, p.o.),
fluoxetine (10 mg/kg, p.o.) or vehicle, rats
were subjected to the FST. Results are shown as
mean ± SEM (n = 6) by vertical bars, where
****
p < 0.0001 (Compared to normal control
group), #p < 0.05, ####p < 0.0001
(Compared to vehicle-treated CUMS-exposed
rats), Two-way analysis of variance (ANOVA),
followed by post-hoc Tukey’s multiple com-
parison test. CUMS, chronic unpredictable mild
stress; CPE, methanolic extract of areal part of
Convolvulus pluricaulis; FST, forced swimming
test.
G.L. Gupta, J. Fernandes
Fig. 6. Influences of CUMS and CPE on pro-inflammatory cytokines in the blood
plasma. (6 A) IL-1β levels, (6B) IL-6 levels (6C) TNF-α levels. Results are shown
as mean ± SEM (n = 6) by vertical bars, where ***p < 0.001, ****p <
0.0001 (Compared to normal control group), #p < 0.05, ##p < 0.01
###
p < 0.001, ####p < 0.0001 (Compared to vehicle-treated CUMS-ex-
posed rats), Two-way analysis of variance (ANOVA), followed by post-hoc
Tukey’s multiple comparison test. CUMS, chronic unpredictable mild stress;
CPE, methanolic extract of areal part of Convolvulus pluricaulis; IL-1β, inter-
leukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α.
3.9. Influences of CPE on the serotonin levels in the hippocampus and
prefrontal cortex
As shown in Fig. 8, two-way ANOVA depicted significant differences
among the treatments groups on the serotonin levels in the hippo-
campus [F (5, 25) = 49.69, p < 0.0001], and prefrontal cortex [F (5,
25) = 36.96, p < 0.0001]. Post-hoc Tukey’s multiple comparison test
showed that CUMS-exposed rats exhibited significant decrement in the
serotonin levels in the hippocampus (p < 0.0001; Fig. 8A), and pre-
frontal cortex (p < 0.0001; Fig. 8B) as compared to the normal control
group. One-week CPE (50, and 100 mg/kg, p.o.) or fluoxetine (10 mg/
kg, p.o.) administration improved the level of serotonin in the hippo-
campus (p < 0.01, p < 0.0001, p < 0.0001; Fig. 8A) as well as in the
prefrontal cortex (p < 0.01, p < 0.0001, p < 0.0001; Fig. 8B) re-
spectively in the CUMS-exposed rats as compared to the vehicle-treated
CUMS-exposed rats, therefore demonstrates the antidepressant-like
activity.
3.10. Influences of CPE on the noradrenaline levels in the hippocampus and
prefrontal cortex
As presented in Fig. 9, two-way ANOVA revealed significant dif-
ferences among the treatments groups on the noradrenaline levels in
the hippocampus [F (5, 25) = 55.95, p < 0.0001], and prefrontal
cortex [F (5, 25) = 22.77, p < 0.0001]. Post-hoc Tukey’s multiple
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Biomedicine & Pharmacotherapy 109 (2019) 1698–1708
comparison test exerted that CUMS-exposed rats demonstrated sig-
nificant decline in the noradrenaline levels in the hippocampus (p <
0.0001; Fig. 9A), and prefrontal cortex (p < 0.0001; Fig. 9B) as com-
pared to the normal control group. One-week CPE (50, and 100 mg/kg,
p.o.) or fluoxetine (10 mg/kg, p.o.) administration reversed the level of
noradrenaline in the hippocampus (p < 0.05, p < 0.0001, p < 0.001;
Fig. 9A) as well as in the prefrontal cortex (p < 0.05, p < 0.0001, p <
0.05; Fig. 9B) respectively in the CUMS-exposed rats as compared to the
vehicle-treated CUMS-exposed rats, therefore exerts the antidepressant-
like effects.
4. Discussion
In the current study, we assessed the antidepressant-like effect of
CPE and also investigated the possible mechanism using a CUMS ex-
posure rat model of depression. Our major findings reveal that CUMS
resulted in increased depression-like behavior, inflammation, liver
biomarkers and decreased serotonin, noradrenaline level in the hippo-
campus as well as in the prefrontal cortex of the rats. CPE-treatment
efficiently ameliorated CUMS-induced depression, pro-inflammatory
cytokines, liver biomarkers, and brain monoaminergic neuro-
transmitters.
The CUMS model is an acclaimed and authentic animal model of
depression with high translational potential because this model can
mimic the core symptoms of the depression by exposing animals to
diverse kind of stress every day [52,63]. Hence, we selected it to re-
search the antidepressant-like effects of CPE in this study. However, the
influence of CPE on depression-like behaviors have been investigated
earlier in other animal models [37].
Moreover, our results revealed that rats exposed to CUMS for 27
consecutive days, demonstrated a remarkable decrease in the sucrose
intake in the SPT, immobility time in the FST, a number of line cross-
ings, rearing in the OFT, locomotor index in the actophotometer, and
regarded as induction of depressive-like behavior in rats. In the earlier
reports, the SPT is used to demonstrate the anhedonia-like behavior in
animals (a decrease of interest to rewards) and, considered as a core
symptom of depression [47,52,64]. In the SPT, rats were given access to
a highly preferred sucrose solution, or to a choice between a sucrose
solution and drinking water. Sucrose preference decreases over weeks
of CUMS exposure but can be restored to normal levels by chronic
treatment with antidepressant drugs [5,15]. Furthermore, we found
that one-week consecutive CPE (50 and 100 mg/kg) or fluoxetine
(10 mg/kg) treatment significantly increased sucrose preference index
and effectively attenuated the depressive-like behavior of CUMS-ex-
posed rats.
The FST was used to evaluate the antidepressant-like effect of the
drugs because it is a reliable animal model with high predictive validity
[59–61]. In the present study, we observed an increment of the im-
mobility time in the FST in the CUMS-exposed rats. It is well reported
earlier that rodents exposed to CUMS display despair behavior and our
results are also consistent with the previous studies [15,65]. One-week
CPE (50 and 100 mg/kg) or fluoxetine (10 mg/kg) treatment sig-
nificantly declined immobility time in the FST, indicating anti-
depressant-like effects of CPE in the CUMS animal model, whereas
25 mg/kg of CPE did not alter the CUMS-induced increase in the im-
mobility time in the FST. Our results are in concordance with earlier
reports which reveal that CPE exerts an antidepressant effect in other
animal models of depression [37]. Therefore, the results confirmed the
antidepressant-like activity of CPE in the CUMS animal model.
We, further, confirmed that whether the decreased immobility in
the FST on the administration of CPE was presented by the anti-
depressant-like effect or the locomotor-stimulant activity. Hence we
used the OFT and actophotometer animal models to evaluate the lo-
comotor, exploratory behaviors [53,54]. In this study, CUMS-exposed
rats presented significant impairment in the number of crossing, rearing
in the OFT and locomotor count in the actophotometer. Treating CUMS-
G.L. Gupta, J. Fernandes
Fig. 8. Effects of CUMS and CPE on the serotonin level in the (8 A) hippo-
campus, and (8B) prefrontal cortex in the CUMS-exposed rats. Results are
shown as mean ± SEM (n = 6) by vertical bars, where ****p < 0.0001
(Compared to normal control group), ##p < 0.01, ####p < 0.0001
(Compared to vehicle-treated CUMS-exposed rats), Two-way analysis of var-
iance (ANOVA), followed by post-hoc Tukey’s multiple comparison test. CUMS,
chronic unpredictable mild stress; CPE, methanolic extract of areal part of
Convolvulus pluricaulis.
exposed rats with CPE (50 and 100 mg/kg) or fluoxetine (10 mg/kg)
restored their locomotion, as evident from the significantly higher
number of line crossings, rearing in the OFT and remarkable restoration
of the locomotion count in the actophotometer. However, in normal
control rats, one-week treatment with all doses of CPE did not alter the
locomotor activity compared to the vehicle-treated normal control
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Biomedicine & Pharmacotherapy 109 (2019) 1698–1708
Fig. 7. Influences of CUMS and CPE on liver
biomarkers in the blood plasma. (7 A) ALT le-
vels, (7B) AST levels (7C) ALP levels. Results
are shown as mean ± SEM (n = 6) by vertical
bars, where **p < 0.01, ***p < 0.001
(Compared to normal control group), #p <
0.05, ##p < 0.01 (Compared to vehicle-
treated CUMS-exposed rats), Two-way analysis
of variance (ANOVA), followed by post-hoc
Tukey’s multiple comparison test. CUMS,
chronic unpredictable mild stress; CPE, me-
thanolic extract of areal part of Convolvulus
pluricaulis; ALT, Alanine transaminase; AST,
Aspartate transaminase, and ALP, Alkaline
phosphatase.
group, suggesting that CPE administration have no influences on the
locomotor activity in rats. These results are in accordance with the
previous studies implying that the antidepressant-like effect of CPE may
be uninvolved by the locomotor-stimulant activity [66].
Interestingly, previous studies have indicated that chronic stress
plays a crucial role in the progression of depression, associated in-
flammation, and disturbance of brain neurotransmitters [20,63,67,68].
Numerous investigations have confirmed that increased inflammatory
cytokines in the brain played a crucial role in the pathogenesis of de-
pression [20,63,69–72]. Therefore, pro-inflammatory cytokines might
be the apparent target for the treatment of depression. Previous studies
have reported that the cytokine-induced behavioral alterations are as-
sociated with changes in the metabolism of serotonin levels in the
hippocampus and prefrontal cortex [4,20,26,71,73,74]. Therefore, we
thought to measure the levels of cytokines and serotonin in the CUMS-
exposed rats, and following treatment with CPE and fluoxetine. Our
results revealed that CUMS markedly aggravated pro-inflammatory
markers IL-1β, IL-6, TNF-α, and depression-like behavior. These find-
ings are consistent with those analogous results obtained after similar
paradigms of CUMS [75]. Clinical studies also revealed that pro-in-
flammatory cytokines, including IL-1β, IL-6, and TNF-α were sig-
nificantly elevated during the depression [76–78]. To further elucidate
the pharmacological mechanism of CPE, the current investigation
probed that one-week treatment of CPE could reverse the inflammatory
cytokines levels induced by the CUMS exposure. These results revealed
that CPE (50 and 100 mg/kg) or fluoxetine (10 mg/kg) exerted an anti-
inflammatory and neuroprotective effect. Interestingly, CPE treatment
at 100 mg/kg completely reversed the CUMS-induced increase in the IL-
1β, IL-6, and TNF-α levels as compared with CUMS-exposed rats. This is
the first report for the action of CPE to ameliorate the pro-inflammatory
cytokines, including IL-1β, IL-6, and TNF-α levels in the CUMS asso-
ciated inflammatory diseases. However, the effects of CPE on the
CUMS-induced depression-like behavior and altered cytokines have
been unscreened yet. We speculated that CPE also inhibited in-
flammation in the CUMS rats via modulating pro-inflammatory
G.L. Gupta, J. Fernandes
Fig. 9. Effects of CUMS and CPE on the noradrenaline level in the (9 A) hip-
pocampus, and (9B) prefrontal cortex in the CUMS-exposed rats. Results are
shown as mean ± SEM (n = 6) by vertical bars, where ****p < 0.0001
(Compared to normal control group), #p < 0.05, ###p < 0.001, ####p <
0.0001 (Compared to vehicle-treated CUMS-exposed rats), Two-way analysis of
variance (ANOVA), followed by post-hoc Tukey’s multiple comparison test.
CUMS, chronic unpredictable mild stress; CPE, methanolic extract of areal part
of Convolvulus pluricaulis.
cytokines associated with serotonin and noradrenaline decline. IL-1β,
IL-6, and TNF-α are the most widely investigated cytokines as a potent
mediator of inflammation and associated depression. The involvement
of IL-1β, IL-6, and TNF-α cytokines to ingress the brain via leaky re-
gions in the blood-brain barrier has been demonstrated earlier in the
pathogenesis of the depression [20,70,71]. These cytokines also dys-
regulated the metabolism of relevant neurotransmitters like serotonin,
noradrenaline, and disruption of synaptic plasticity through alterations
brain-derived neurotrophic factor. However, activation of the kynur-
enine pathway of tryptophan metabolism may be involved in the pa-
thogenesis of the depression (Bryleva and Brundin, 2016; O'Connor
et al., 2009).
Increasing evidence suggested that monoaminergic deficiency has
been regarded to underlie depressive disorders and most of the existing
antidepressants work by increasing the serotonin and/or noradrenaline
levels [79–81]. In the present study, we also observed a significant
decline of serotonin and noradrenaline levels in the hippocampus as
well as in the prefrontal cortex of the CUMS-exposed rats. Further, one
week CPE (50 and 100 mg/kg) or fluoxetine (10 mg/kg) treatment
significantly reversed the serotonin and noradrenaline levels in the
hippocampus as well as in the prefrontal cortex of the CUMS-exposed
rats, stipulating that the antidepressant-like effect of CPE could be due
to the improvement of major monoamine neurotransmitters serotonin
and noradrenaline, these results are also consistent with the previous
reports [37]. The results of CPE were found to be comparable with that
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Biomedicine & Pharmacotherapy 109 (2019) 1698–1708
of the standard drug fluoxetine.
On other way, studies on depression are mainly engrossed on the
changes in the brain, but not on the liver. On a primary level, we also
measured whether CUMS alter the liver enzymes and also screening the
influences of CPE in the altered liver biomarkers. In our study, one-
week CPE (100 mg/kg) or fluoxetine (10 mg/kg) treatments sig-
nificantly declined the increased levels of liver enzymes ALT and AST
induced by CUMS, indicating CPE was capable of ameliorating the
hepatic injury. Our finding also supports the previous reports on the
elevation of liver enzymes during CUMS [5,82]. It was also suggested
that depression could be linked with liver injury in the earlier reports
[5,83,84], and therefore, CPE can also ameliorate depressive-like be-
havior by inhibiting liver-brain inflammation axis.
5. Conclusions
Based on our results, we conclude that CUMS exposure instigated
depression-like behavior as well as alteration in inflammatory cyto-
kines, liver enzymes, serotonin and noradrenaline levels.
Administration of CPE attenuated CUMS-induced depressive-like be-
havior. The antidepressant-like effect of CPE in the CUMS-exposed rats
may be linked to the alteration of inflammatory cytokines, liver en-
zymes, serotonin and noradrenaline levels. CPE may be a potential drug
for the treatment of depression and its associated neuroinflammation.
However, further studies are needed to explore the gene and protein
expression alteration to understand the molecular mechanism of CPE
effects in CUMS-induced depression.
Conflict of interest
The authors declare no conflict of interest.
Funding information
Authors are grateful to SVKM’s NMIMS (Deemed to be University),
Mumbai, India for providing a grant for the research.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.biopha.2018.11.046.
References
[1] X.L. Zhu, J.J. Chen, F. Han, C. Pan, T.T. Zhuang, Y.F. Cai, Y.P. Lu, Novel anti-
depressant effects of Paeonol alleviate neuronal injury with concomitant alterations
in BDNF, Rac1 and RhoA levels in chronic unpredictable mild stress rats,
Psychopharmacology (Berl.) (2018) 1–15, https://doi.org/10.1007/s00213-018-
4915-7.
[2] World Health Organization, Depression and other common mental disorders: global
health estimates, World Heal. Organ. (2017) 1–24 doi:CC BY-NC-SA 3.0 IGO.
[3] N. Eshel, J.P. Roiser, Reward and punishment processing in depression, Biol.
Psychiatry 68 (2010) 118–124, https://doi.org/10.1016/j.biopsych.2010.01.027.
[4] Z.Q. Li, Z.Y. Yan, F.J. Lan, Y.Q. Dong, Y. Xiong, Suppression of NLRP3 inflamma-
some attenuates stress-induced depression-like behavior in NLGN3-deficient mice,
Biochem. Biophys. Res. Commun. 501 (2018) 933–940, https://doi.org/10.1016/j.
bbrc.2018.05.085.
[5] H.M. Jia, Q. Li, C. Zhou, M. Yu, Y. Yang, H.W. Zhang, G. Ding, H. Shang, Z.M. Zou,
Chronic unpredictive mild stress leads to altered hepatic metabolic profile and gene
expression, Sci. Rep. 6 (2016) 1–10, https://doi.org/10.1038/srep23441.
[6] D.M. Kokare, P.S. Singru, M.P. Dandekar, C.T. Chopde, N.K. Subhedar, Involvement
of alpha-melanocyte stimulating hormone (α-MSH) in differential ethanol exposure
and withdrawal related depression in rat: neuroanatomical-behavioral correlates,
Brain Res. 1216 (2008) 53–67, https://doi.org/10.1016/j.brainres.2008.03.064.
[7] V. Krishnan, E.J. Nestler, The molecular neurobiology of depression, Nature 455
(2008) 894–902, https://doi.org/10.1038/nature07455.
[8] K. Yan, Y.-B. Chen, J.-R. Wu, K.-D. Li, Y.-L. Cui, Current rapid-onset antidepressants
and related animal models, Curr. Pharm. Des. 24 (2018), https://doi.org/10.2174/
1381612824666180727115222.
[9] D.M. Gerhard, E.S. Wohleb, R.S. Duman, Emerging treatment mechanisms for de-
pression: focus on glutamate and synaptic plasticity, Drug Discov. Today 21 (2016)
454–464, https://doi.org/10.1016/j.drudis.2016.01.016.
G.L. Gupta, J. Fernandes
[10] D.M. Gerhard, R.S. Duman, Rapid-acting antidepressants: mechanistic insights and
future directions, Curr. Behav. Neurosci. Reports. 5 (2018) 36–47, https://doi.org/
10.1016/j.chemosphere.2012.12.037.Reactivity.
[11] J.P. Pandarakalam, Challenges of treatment-resistant depression, Psychiatr. Danub.
30 (2018) 273–284, https://doi.org/10.24869/psyd.2018.273.
[12] R. Chockalingam, B.M. Gott, C.R. Conway, Tricyclic Antidepressants and
Monoamine Oxidase Inhibitors: Are They Too Old for a New Look? (2018), https://
doi.org/10.1007/164_2018_133.
[13] T.C. Lee, A.M. Alessio, R.M. Miyaoka, P.E. Kinahan, Morphology supporting func-
tion: Attenuation correction for SPECT/CT, PET/CT, and PET/MR imaging, Q. J.
Nucl. Med. Mol. Imaging 60 (2016) 25–39, https://doi.org/10.1016/j.
chemosphere.2012.12.037.Reactivity.
[14] J.W. Murrough, C.G. Abdallah, S.J. Mathew, Targeting glutamate signalling in
depression: progress and prospects, Nat. Rev. Drug Discov. 16 (2017) 472–486,
https://doi.org/10.1038/nrd.2017.16.
[15] T. Qin, F. Fang, M. Song, R. Li, Z. Ma, S. Ma, Umbelliferone reverses depression-like
behavior in chronic unpredictable mild stress-induced rats by attenuating neuronal
apoptosis via regulating ROCK/Akt pathway, Behav. Brain Res. 317 (2017)
147–156, https://doi.org/10.1016/j.bbr.2016.09.039.
[16] M. Berk, L.J. Williams, F.N. Jacka, A.O. Neil, J.A. Pasco, S. Moylan, N.B. Allen,
A.L. Stuart, A.C. Hayley, M.L. Byrne, M. Maes, So depression is an inflammatory
disease, but where does the inflammation come from? BMC Med. 11 (2013) 200,
https://doi.org/10.1186/1741-7015-11-200.
[17] S.Q. Dong, Q.P. Zhang, J.X. Zhu, M. Chen, C.F. Li, Q. Liu, D. Geng, L.T. Yi,
Gypenosides reverses depressive behavior via inhibiting hippocampal neuroin-
flammation, Biomed. Pharmacother. 106 (2018) 1153–1160, https://doi.org/10.
1016/j.biopha.2018.07.040.
[18] H. Jiang, G. Zheng, J. Lv, H. Chen, J. Lin, Y. Li, G. Fan, X. Ding, Identification of
Centella asiatica’s effective ingredients for inducing the neuronal differentiation,
evidence-based complement, Altern. Med. 2016 (2016), https://doi.org/10.1155/
2016/9634750.
[19] C. Fan, Q. Song, P. Wang, Y. Li, M. Yang, B. Liu, S.Y. Yu, Curcumin protects against
chronic stress-induced dysregulation of neuroplasticity and depression-like beha-
viors via suppressing IL-1β pathway in rats, Neuroscience (2018), https://doi.org/
10.1016/j.neuroscience.2018.09.028.
[20] C.L. Raison, L. Capuron, A.H. Miller, Cytokines sing the blues: inflammation and the
pathogenesis of depression, Trends Immunol. 27 (2006) 24–31, https://doi.org/10.
1016/j.it.2005.11.006.
[21] Y.M. Liu, J.D. Shen, L.P. Xu, H.B. Li, Y.C. Li, L.T. Yi, Ferulic acid inhibits neuro-
inflammation in mice exposed to chronic unpredictable mild stress, Int.
Immunopharmacol. 45 (2017) 128–134, https://doi.org/10.1016/j.intimp.2017.
02.007.
[22] J.C. Felger, Z. Li, E. Haroon, B.J. Woolwine, M.Y. Jung, X. Hu, A.H. Miller,
Inflammation is associated with decreased functional connectivity within corti-
costriatal reward circuitry in depression, Mol. Psychiatry 21 (2016) 1358–1365,
https://doi.org/10.1038/mp.2015.168.
[23] C. Yang, F.J. Bosker, J. Li, R.A. Schoevers, N-acetylcysteine as add-on to anti-
depressant medication in therapy refractory major depressive disorder patients with
increased inflammatory activity: Study protocol of a double-blind randomized
placebo-controlled trial, BMC Psychiatry 18 (2018) 279, https://doi.org/10.1186/
s12888-018-1845-1.
[24] F.P. MacMaster, N. Carrey, L.M. Langevin, N. Jaworska, S. Crawford, Disorder-
specific volumetric brain difference in adolescent major depressive disorder and
bipolar depression, Brain Imaging Behav. 8 (2014) 119–127, https://doi.org/10.
1007/s11682-013-9264-x.
[25] W.-Y. Zhang, K.-Y. Wang, Y.-J. Li, Y.-R. Li, R.-Z. Lu, Chronic stress causes protein
kinase C epsilon-aldehyde dehydrogenase 2 signaling pathway perturbation in the
rat hippocampus and prefrontal cortex, but not in the myocardium, Neural Regen.
Res. 13 (2018) 1225–1230, https://doi.org/10.4103/1673-5374.235060.
[26] Y. Zhang, K. Fan, Y. Liu, G. Liu, X. Yang, J. Ma, Cathepsin C aggravates neuroin-
flammation involved in disturbances of behaviour and neurochemistry in acute and
chronic stress-induced murine model of depression, Neurochem. Res. 43 (2018)
1702, https://doi.org/10.1007/s11064-018-2564-1.
[27] K. Sharma, M. Bhatnagar, S.K. Kulkarni, Effect of Convolvulus pluricaulis Choisy
and Asparagus racemosus Willd on learning and memory in young and old mice: a
comparative evaluation, Indian J. Exp. Biol. 48 (2010) 479–485 (Accessed 19
October 2018), http://www.ncbi.nlm.nih.gov/pubmed/20795365.
[28] H. Amin, R. Sharma, P. Prajapati, R. Dwivedi, H. Vyas, M. Vyas, Nootropic
(medhya) effect of Bhāvita Śaṇkhapuṣpī tablets: a clinical appraisal, Anc. Sci. Life
34 (2014) 109–112, https://doi.org/10.4103/0257-7941.153476.
[29] P.A. Kizhakke, S. Olakkaran, A. Antony, K.S. Tilagul, P.G. Hunasanahally,
Convolvulus pluricaulis (Shankhapushpi) ameliorates human microtubule-asso-
ciated protein tau (hMAPτ) induced neurotoxicity in Alzheimer’s disease Drosophila
model, J. Chem. Neuroanat. (2017), https://doi.org/10.1016/j.jchemneu.2017.10.
002.
[30] S. Bihaqi, A. Singh, M. Tiwari, Supplementation of Convolvulus pluricaulis at-
tenuates scopolamine-induced increased tau and Amyloid precursor protein (AβPP)
expression in rat brain, Indian J. Pharmacol. 44 (2012) 593–598, https://doi.org/
10.4103/0253-7613.100383.
[31] S.W. Bihaqi, M. Sharma, A.P. Singh, M. Tiwari, Neuroprotective role of Convolvulus
pluricaulis on aluminium induced neurotoxicity in rat brain, J. Ethnopharmacol.
124 (2009) 409–415, https://doi.org/10.1016/j.jep.2009.05.038.
[32] M. Kaur, A. Prakash, A.N. Kalia, Neuroprotective potential of antioxidant potent
fractions from Convolvulus pluricaulis Chois. In 3-nitropropionic acid challenged
rats, Nutr. Neurosci. 19 (2016) 70–78, https://doi.org/10.1179/1476830515Y.
0000000022.
1707
Biomedicine & Pharmacotherapy 109 (2019) 1698–1708
[33] P. Rachitha, K. Krupashree, G.V. Jayashree, H.K. Kandikattu, N. Amruta,
N. Gopalan, M.K. Rao, F. Khanum, Chemical composition, antioxidant potential,
macromolecule damage and neuroprotective activity of Convolvulus pluricaulis, J.
Tradit. Complement. Med. 8 (2018) 483–496, https://doi.org/10.1016/j.jtcme.
2017.11.002.
[34] J. Malik, M. Karan, K. Vasisht, Attenuating effect of bioactive coumarins from
Convolvulus pluricaulis on scopolamine-induced amnesia in mice, Nat. Prod. Res.
30 (2016) 578–582, https://doi.org/10.1080/14786419.2015.1025398.
[35] J. Malik, S. Choudhary, P. Kumar, Protective effect of Convolvulus pluricaulis
standardized extract and its fractions against 3-nitropropionic acid-induced neu-
rotoxicity in rats, Pharm. Biol. 53 (2015) 1448–1457, https://doi.org/10.3109/
13880209.2014.984856.
[36] M. Heba, S. Faraz, S. Banerjee, Effect of Shankhpushpi on alcohol addiction in mice,
Pharmacogn. Mag. 13 (2017) S148–S153, https://doi.org/10.4103/0973-1296.
203976.
[37] D. Dhingra, R. Valecha, Evaluation of the antidepressant-like activity of
Convolvulus pluricaulis choisy in the mouse forced swim and tail suspension tests,
Med. Sci. Monit. 13 (2007) BR155–61 http://www.ncbi.nlm.nih.gov/pubmed/
17599020.
[38] G. Garg, A. Patil, J. Singh, N. Kaushik, A. Praksah, A. Pal, A. Chakrabarti,
Pharmacological evaluation of Convolvulus pluricaulis as hypolipidaemic agent in
Triton WR-1339-induced hyperlipidaemia in rats, J. Pharm. Pharmacol. 70 (2018)
1572–1580, https://doi.org/10.1111/jphp.13004.
[39] Sristi Verma, Reema Sinha, Puspendra Kumar, Faizal Amin, Jainendra Jain,
Shivani Tanwar, Study of Convolvulus pluricaulis for antioxidant and antic-
onvulsant activity, Cent. Nerv. Syst. Agents Med. Chem. 12 (2012) 55–59, https://
doi.org/10.2174/187152412800229161.
[40] A. Dhar, S.K. Maurya, A. Mishra, G.K. Singh, M.K. Singh, A. Seth, Preliminary
screening of a classical ayurvedic formulation for anticonvulsant activity, Anc. Sci.
Life 36 (2016) 28–34, https://doi.org/10.4103/0257-7941.195410.
[41] A. Nahata, U.K. Patil, V.K. Dixit, Anxiolytic activity of Evolvulus alsinoides and
Convulvulus pluricaulis in rodents, Pharm. Biol. 47 (2009) 444–451, https://doi.org/
10.1080/13880200902822596.
[42] K. Sairam, C.V. Rao, R.K. Goel, Effect of Convolvulus pluricaulis Chois on gastric
ulceration and secretion in rats, Indian J. Exp. Biol. 39 (2001) 350–354 (accessed
September 20, 2018), http://www.ncbi.nlm.nih.gov/pubmed/11491580.
[43] N.K. Sethiya, S. Mishra, Review on ethnomedicinal uses and phyto-pharmacology of
memory boosting herb Convolvulus pluricaulis Choisy, Aust. J. Med. Herbal. 22
(2010) 19–25 http://www.encognitive.com/files/Reviewonethnomedicinalusesand
phytopharmacologyofmemoryboostingherbConvolvuluspluricaulisChoisy.pdf.
[44] P. Agarwal, B. Sharma, A. Fatima, S.K. Jain, An update on Ayurvedic Herb
Convolvulus pluricaulis Choisy, Asian Pac. J. Trop. Biomed. 4 (2014) 245–252,
https://doi.org/10.1016/S2221-1691(14)60240-9.
[45] J.C. Capra, M.P. Cunha, D.G. Machado, A.D.E. Zomkowski, B.G. Mendes,
A.R.S. Santos, M.G. Pizzolatti, A.L.S. Rodrigues, Antidepressant-like effect of sco-
poletin, a coumarin isolated from Polygala sabulosa (Polygalaceae) in mice: evi-
dence for the involvement of monoaminergic systems, Eur. J. Pharmacol. 643
(2010) 232–238, https://doi.org/10.1016/j.ejphar.2010.06.043.
[46] H.-J. Kim, S. Il Jang, Y.-J. Kim, H.-T. Chung, Y.-G. Yun, T.-H. Kang, O.-S. Jeong, Y.-
C. Kim, Scopoletin suppresses pro-inflammatory cytokines and PGE2 from LPS-sti-
mulated cell line, RAW 264.7 cells, Fitoterapia 75 (2004) 261–266, https://doi.org/
10.1016/j.fitote.2003.12.021.
[47] P. Willner, The chronic mild stress (CMS) model of depression: history, evaluation
and usage, Neurobiol. Stress 6 (2017) 78–93, https://doi.org/10.1016/j.ynstr.2016.
08.002.
[48] W.C. Evans, D. Evans, G.E. Trease, Trease and Evans Pharmacognosy, Saunders/
Elsevier, 2009.
[49] H. Amin, R. Sharma, M. Vyas, P.K. Prajapati, K. Dhiman, Shankhapushpi
(Convolvulus pluricaulis Choisy): validation of the Ayurvedic therapeutic claims
through contemporary studies, Int. J. Green Pharm. (2014) 193–200, https://doi.
org/10.4103/0973-8258.142666.
[50] OECD, Guideline 423. Acute Oral Toxicity- Acute Toxic Class Method. 470 Adopted
by the Council on 17th, December 2001, 2001, Organization for economic co-
operation and development (OECD), 2001 (accessed October 1, 2018), https://ntp.
niehs.nih.gov/iccvam/suppdocs/feddocs/oecd/oecd_gl423.pdf.
[51] D.G. MacHado, M.P. Cunha, V.B. Neis, G.O. Balen, A. Colla, J. Grando,
P.S. Brocardo, L.E.B. Bettio, J.C. Capra, A.L.S. Rodrigues, Fluoxetine reverses de-
pressive-like behaviors and increases hippocampal acetylcholinesterase activity
induced by olfactory bulbectomy, Pharmacol. Biochem. Behav. 103 (2012)
220–229, https://doi.org/10.1016/j.pbb.2012.08.024.
[52] G.L. Wang, Y.P. Wang, J.Y. Zheng, L.X. Zhang, Monoaminergic and aminoacidergic
receptors are involved in the antidepressant-like effect of ginsenoside Rb1 in mouse
hippocampus (CA3) and prefrontal cortex, Brain Res. 1699 (2018) 44–53, https://
doi.org/10.1016/j.brainres.2018.05.035.
[53] X. Zu, M. Zhang, W. Li, H. Xie, Z. Lin, N. Yang, X. Liu, W. Zhang, Antidepressant-like
effect of Bacopaside I in mice exposed to chronic unpredictable mild stress by
modulating the hypothalamic–pituitary–adrenal axis function and activating BDNF
signaling pathway, Neurochem. Res. 42 (2017) 3233–3244, https://doi.org/10.
1007/s11064-017-2360-3.
[54] L. Cai, R. Li, W.J. Tang, G. Meng, X.Y. Hu, T.N. Wu, Antidepressant-like effect of
geniposide on chronic unpredictable mild stress-induced depressive rats by reg-
ulating the hypothalamus-pituitary-adrenal axis, Eur. Neuropsychopharmacol. 25
(2015) 1332–1341, https://doi.org/10.1016/j.euroneuro.2015.04.009.
[55] J.F. Ge, W.C. Gao, W.M. Cheng, W.L. Lu, J. Tang, L. Peng, N. Li, F.H. Chen, Orcinol
glucoside produces antidepressant effects by blocking the behavioural and neuronal
deficits caused by chronic stress, Eur. Neuropsychopharmacol. 24 (2014) 172–180,
G.L. Gupta, J. Fernandes
https://doi.org/10.1016/j.euroneuro.2013.05.007.
[56] W. Song, Y. Guo, S. Jiang, L. Wei, Z. Liu, X. Wang, Y. Su, Antidepressant effects of
the Ginsenoside metabolite compound K, assessed by behavioral despair test and
chronic unpredictable mild stress model, Neurochem. Res. 43 (2018) 1371–1382,
https://doi.org/10.1007/s11064-018-2552-5.
[57] A. Kumar, A. Prakash, D. Pahwa, Galantamine potentiates the protective effect of
rofecoxib and caffeic acid against intrahippocampal Kainic acid-induced cognitive
dysfunction in rat, Brain Res. Bull. 85 (2011) 158–168, https://doi.org/10.1016/j.
brainresbull.2011.03.010.
[58] M. Erburu, I. Muñoz-Cobo, T. Diaz-Perdigon, P. Mellini, T. Suzuki, E. Puerta,
R.M. Tordera, SIRT2 inhibition modulate glutamate and serotonin systems in the
prefrontal cortex and induces antidepressant-like action, Neuropharmacology 117
(2017) 195–208, https://doi.org/10.1016/j.neuropharm.2017.01.033.
[59] D.M. Kokare, M.P. Dandekar, P.S. Singru, G.L. Gupta, N.K. Subhedar, Involvement
of α-MSH in the social isolation induced anxiety- and depression-like behaviors in
rat, Neuropharmacology 58 (2010) 1009–1018, https://doi.org/10.1016/j.
neuropharm.2010.01.006.
[60] R. Yawalkar, H. Changotra, G.L. Gupta, Protective influences of N-acetylcysteine
against alcohol abstinence-induced depression by regulating biochemical and
GRIN2A, GRIN2B gene expression of NMDA receptor signaling pathway in rats,
Neurochem. Int. 118 (2018) 73–81, https://doi.org/10.1016/j.neuint.2018.04.011.
[61] R.D. Porsolt, G. Anton, N. Blavet, M. Jalfre, Behavioural despair in rats: a new
model sensitive to antidepressant treatments, Eur. J. Pharmacol. 47 (1978)
379–391, https://doi.org/10.1016/0014-2999(78)90118-8.
[62] J. Thomas, R. Khanam, D. Vohora, A validated HPLC-UV method and optimization
of sample preparation technique for norepinephrine and serotonin in mouse brain,
Pharm. Biol. 53 (2015) 1539–1544, https://doi.org/10.3109/13880209.2014.
991837.
[63] C. Zhang, Y.P. Zhang, Y.Y. Li, B.P. Liu, H.Y. Wang, K.W. Li, S. Zhao, C. Song,
Minocycline ameliorates depressive behaviors and neuro-immune dysfunction in-
duced by chronic unpredictable mild stress in the rat, Behav. Brain Res. 356 (2019)
348–357, https://doi.org/10.1016/j.bbr.2018.07.001.
[64] J. Xue, H. Li, X. Deng, Z. Ma, Q. Fu, S. Ma, L-Menthone confers antidepressant-like
effects in an unpredictable chronic mild stress mouse model via NLRP3 in-
flammasome-mediated inflammatory cytokines and central neurotransmitters,
Pharmacol. Biochem. Behav. 134 (2015) 42–48, https://doi.org/10.1016/j.pbb.
2015.04.014.
[65] Y. Song, R. Sun, Z. Ji, X. Li, Q. Fu, S. Ma, Perilla aldehyde attenuates CUMS-induced
depressive-like behaviors via regulating TXNIP/TRX/NLRP3 pathway in rats, Life
Sci. 206 (2018) 117–124, https://doi.org/10.1016/j.lfs.2018.05.038.
[66] N.A. Siddiqui, N. Ahmad, N. Musthaq, I. Chattopadhyaya, R. Kumria, S. Gupta,
Neuropharmacological profile of extracts of aerial parts of Convolvulus pluricaulis
Choisy in mice model, Open Neurol. J. 8 (2014) 11–14, https://doi.org/10.2174/
1874205X01408010011.
[67] H. Gao, X. Zhu, Y. Xi, Q. Li, Z. Shen, Y. Yang, Anti-depressant-like effect of atrac-
tylenolide I in a mouse model of depression induced by chronic unpredictable mild
stress, Exp. Ther. Med. (2017) 1574–1579, https://doi.org/10.3892/etm.2017.
5517.
[68] V.S. Velingkar, G.L. Gupta, N.B. Hegde, A current update on phytochemistry,
pharmacology and herb–drug interactions of Hypericum perforatum, Phytochem.
Rev. 16 (2017) 725–744, https://doi.org/10.1007/s11101-017-9503-7.
[69] C. Park, E. Brietzke, J.D. Rosenblat, N. Musial, H. Zuckerman, R.M. Ragguett,
Z. Pan, C. Rong, D. Fus, R.S. McIntyre, Probiotics for the treatment of depressive
symptoms: An anti-inflammatory mechanism? Brain Behav. Immun. 73 (2018)
115–124, https://doi.org/10.1016/j.bbi.2018.07.006.
[70] N. Lichtblau, F.M. Schmidt, R. Schumann, K.C. Kirkby, H. Himmerich, Cytokines as
biomarkers in depressive disorder: current standing and prospects, Int. Rev.
Psychiatry 25 (2013) 592–603, https://doi.org/10.3109/09540261.2013.813442.
1708
Biomedicine & Pharmacotherapy 109 (2019) 1698–1708
[71] J.D. Rosenblat, J.M. Gregory, R.S. McIntyre, Pharmacologic implications of in-
flammatory comorbidity in bipolar disorder, Curr. Opin. Pharmacol. 29 (2016)
63–69, https://doi.org/10.1016/j.coph.2016.06.007.
[72] R. Haapakoski, J. Mathieu, K.P. Ebmeier, H. Alenius, M. Kivimäki, Cumulative
meta-analysis of interleukins 6 and 1β, tumour necrosis factor α and C-reactive
protein in patients with major depressive disorder, Brain Behav. Immun. 49 (2015)
206–215, https://doi.org/10.1016/j.bbi.2015.06.001.
[73] E.A. Rahim, Z. Mohd-Zain, J. Hussaini, E.H. Wong, L.L. Nyein, N. Parasakthi,
A. Adnan, N.K. Palanisamy, Adherence of Streptococcus pneumoniae and expres-
sion analysis of neuraminidase gene (NanA and NanB) after interaction of A549
human lung epithelial cells with pneumococcal strains of various serotypes, Malays.
J. Microbiol. 13 (2017) 210–216, https://doi.org/10.1007/978-0-585-37970-8_8.
[74] B. Sperner-Unterweger, C. Kohl, D. Fuchs, Immune changes and neurotransmitters:
Possible interactions in depression? Prog. Neuro-Psychopharmacol. Biol.
Psychiatry. 48 (2014) 268–276, https://doi.org/10.1016/j.pnpbp.2012.10.006.
[75] X. Liu, C. Liu, Baicalin ameliorates chronic unpredictable mild stress-induced de-
pressive behavior: involving the inhibition of NLRP3 inflammasome activation in
rat prefrontal cortex, Int. Immunopharmacol. 48 (2017) 30–34, https://doi.org/10.
1016/j.intimp.2017.04.019.
[76] P. Martinez, L. Lien, S. Zemore, J.G. Bramness, S.P. Neupane, Circulating cytokine
levels are associated with symptoms of depression and anxiety among people with
alcohol and drug use disorders, J. Neuroimmunol. 318 (2018) 80–86, https://doi.
org/10.1016/j.jneuroim.2018.02.011.
[77] A. Dey, P. Hankey Giblin, Insights into macrophage heterogeneity and cytokine-
induced neuroinflammation in major depressive disorder, Pharmaceuticals 11
(2018) 64, https://doi.org/10.3390/ph11030064.
[78] C.A. Köhler, T.H. Freitas, B. Stubbs, M. Maes, M. Solmi, N. Veronese, N.Q. de
Andrade, G. Morris, B.S. Fernandes, A.R. Brunoni, N. Herrmann, C.L. Raison,
B.J. Miller, K.L. Lanctôt, A.F. Carvalho, Peripheral alterations in cytokine and
chemokine levels after antidepressant drug treatment for major depressive disorder:
systematic review and meta-analysis, Mol. Neurobiol. 55 (2018) 4195–4206,
https://doi.org/10.1007/s12035-017-0632-1.
[79] D.C. Blanchard, K. Meyza, Risk assessment and serotonin: animal models and
human psychopathologies, Behav. Brain Res. (2017), https://doi.org/10.1016/j.
bbr.2017.07.008.
[80] U.E. Lang, S. Borgwardt, Molecular mechanisms of depression: perspectives on new
treatment strategies, Cell. Physiol. Biochem. 31 (2013) 761–777, https://doi.org/
10.1159/000350094.
[81] P. Xu, K.Z. Wang, C. Lu, L.M. Dong, J. Le Zhai, Y.H. Liao, S. Aibai, Y. Yang, X.M. Liu,
Antidepressant-like effects and cognitive enhancement of the total phenols extract
of Hemerocallis citrina Baroni in chronic unpredictable mild stress rats and its re-
lated mechanism, J. Ethnopharmacol. 194 (2016) 819–826, https://doi.org/10.
1016/j.jep.2016.09.023.
[82] H. mei Jia, M. Yu, L.Y. Ma, H. wu Zhang, Z. mei Zou, Chaihu-Shu-Gan-San regulates
phospholipids and bile acid metabolism against hepatic injury induced by chronic
unpredictable stress in rat, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1064
(2017) 14–21, https://doi.org/10.1016/j.jchromb.2017.08.003.
[83] K.-K. Jia, S.-M. Pan, H. Ding, J.-H. Liu, Y.-J. Zheng, S.-J. Wang, Y. Pan, L.-D. Kong,
Chaihu-shugan san inhibits inflammatory response to improve insulin signaling in
liver and prefrontal cortex of CUMS rats with glucose intolerance, Biomed.
Pharmacother. 103 (2018) 1415–1428, https://doi.org/10.1016/j.biopha.2018.04.
171.
[84] K.-K. Jia, Y.-J. Zheng, Y.-X. Zhang, J.-H. Liu, R.-Q. Jiao, Y. Pan, L.-D. Kong, Banxia-
houpu decoction restores glucose intolerance in CUMS rats through improvement of
insulin signaling and suppression of NLRP3 inflammasome activation in liver and
brain, J. Ethnopharmacol. 209 (2017) 219–229, https://doi.org/10.1016/j.jep.
2017.08.004.