Metab Brain Dis (2017) 32:1963–1973

DOI 10.1007/s11011-017-0089-y

ORIGINAL ARTICLE

Morus nigra and its major phenolic, syringic acid,

have antidepressant-like and neuroprotective effects in mice
Ana Paula Dalmagro 1,2 & Anderson Camargo 2 & Ana Lúcia Bertarello Zeni 1,2

Received: 6 February 2017 / Accepted: 11 August 2017 / Published online: 18 August 2017
# Springer Science+Business Media, LLC 2017

Abstract Depression is a disorder with a high incidence Keywords Morus nigra . Antidepressant . Neuprotective .
that has been increasing worldwide although the patho- Syringic acid
physiology remains unclear. Moreover, some studies re-
vealed a higher concentration of glutamate and oxidative
stress in the patients’ brain, which causes cell death by Introduction
excitotoxicity. Morus nigra L. is known as black mulberry
and its leaves are popularly used to treat affections related Depression is a common, recurrent and incapacitating disorder
to menopause, obesity and high cholesterol. M. nigra with a high incidence worldwide (Berton and Nestler 2006).
leaves are a rich fount of phenolics which well-known by Despite the approached efforts, the influence of brain oxidative
the antioxidant property. Herein, we examined the phenolic stress in the illness mechanism has not been entirely explained.
profile and the antidepressant-like effect of the Morus Although, it is established that reactive oxigen species (ROS) is
nigra aqueous extract (MN) and its major phenolic constit- implicated in the depression pathophysiology (Lee et al. 2013;
uent, syringic acid (SA). Furthermore, the involvement of Lucca et al. 2009; Maes et al. 2011), causing damage to DNA,
antioxidant and neuroprotective activities were further lipids and proteins (Pandya et al. 2013). Therefore, leading to
evaluated. Our results show that acute and subchronic cell death and decreasing neurogenesis (Maes et al. 2011). The
MN or SA administration exerted antidepressant-like prop- symptons have been extensively associated with the features of
erty in the behavioral testes in mice. The results suggest an inflammatory response, regarding an increased expression
that the antidepressant-like effect of MN, at least in part, of proinflammatory cytokines and their receptors, including the
could be due to the SA influence. Moreover, the observed raise of chemokines and soluble adhesion molecules levels
effect involves the nitro-oxidative system modulation in (Maes 1999; Miller et al. 2009). Some studies suggest that
both the serum and brain of mice. Furthermore, MN or hypercholesterolemia is also related to depression (Tyrovolas
SA was able to contain the glutamate-induced cell death et al. 2009; Davison and Kaplan 2012). Recently, Engel et al.
in the hippocampal and cortical slices implicating the neu- (2016) demonstrated that hypercholesterolemic mice presented
roprotection activity in the antidepressant-like effect. antidepressant-like effect with alterations in the monoaminergic
system. Previously, Tedders et al. (2011) and Wysokinski et al.
(2015) showed an association between low HDL-cholesterol
level and a severe depression.
* Ana Lúcia Bertarello Zeni The most common treatments for depression are related to
anazeni@furb.br; zeni.ana@gmail.com
the monoaminergic system, moreover, the therapy has also
1
been improved concerning glutamatergic system with the an-
Programa de Pós-Graduação em Química, Departamento de
Química, Universidade Regional de Blumenau, Blumenau, Santa
tagonist of N-Methyl-D-Aspartate (NMDA) receptor, as the
Catarina CEP 89030-903, Brazil ketamine (Sałat et al. 2015). In this regard, post mortem studies
2
Laboratório de Avaliação de Substâncias Bioativas, Departamento de
revealed higher concentration of glutamate in the patient’s brain
Ciências Naturais, Universidade Regional de Blumenau, CEP (Hashimoto et al. 2007) evidencing a link between hyperacti-
89030-903, Campus I, Blumenau, SC 89012-900, Brazil vation of NMDA receptor and cell death by excitotoxicity.
1964 Metab Brain Dis (2017) 32:1963–1973

Morus nigra L. (Moraceae) known as black mulberry is a latitude 26°54′10″ S, longitude 49°04′44″ W). The species
tree distributed worldwide and their fruits are famous due to identified, taxonomically authenticated and the voucher spec-
the high nutritional value (Gundogdu et al. 2011).The leaves imen (N° 42,265) deposited at the Regional University of
are used for popular therapeutical purposes alleviating chronic Blumenau’s herbarium, Santa Catarina, Brazil. The plant ma-
metabolic disorders (Volpato et al. 2011; Miranda et al. 2010). terial dried at 45 °C with forced ventilation, grinded, and then
Moreover, Morus nigra leaves have shown scientific evidence stored at −10 °C.
of anti-inflammatory (Padilha et al. 2010), antinociceptive The preparation of MN was performed using 2 g of
(Padilha et al. 2009) and hepatoprotective (Malhi et al. M. nigra grinded leaves extracted with 100 ml of hot water
2014) effects. In a previous study, we demonstrated that during 10 min, named infusion (Moreira 2015).
M. nigra has lipid-lowering effect in hyperlipidemic rats ac-
companied by the decrement of lipid peroxidation in the brain High performance liquid chromatography (HPLC) profile
(Moreira 2015). In addition, M. nigra did not exert a toxic of MN
effect on the female reproductive system or on the embryonic
development (Volpato et al. 2011; Queiroz et al. 2012). Firstly, the aqueous extract underwent a liquid-liquid extrac-
Finally, Oliveira et al. (2013) have demonstrated that tion with ethyl acetate according to Zeni et al. (2013). The
M. nigra leaf aqueous extract is safe after a 30–day treatment HPLC profile of MN was performed according to Moreira
by oral route in rats. (2015), with modifications. The syringic acid used as standard
Furthermore, some studies indicated that black mulberry leaf for identification was purchased from Sigma-Aldrich,
is a rich source of phenolic compounds which provides a poten- Steinheim, Germany. Preliminary quantification was estimat-
tial antioxidant activity (Araujo et al. 2015; Memon et al. 2010; ed by % area per peak.
Malhi et al. 2014; Sánchez-Salcedo et al. 2015). Also,
polyphenolic-rich extracts or certain phenolic compounds have Animals
demonstrated neuroprotective and antidepressant-like properties
as Aloysia gratissima and ferulic acid (Zeni et al. 2011, 2014; Male Swiss mice (30–40 g) were maintained at 21–23 °C with
Lenzi et al. 2015). Aditionally, other authors were able to dem- free access to water and food, under a 12:12 h light/dark cycle
onstrate these same properties mentioned previously with (lights-on at 07:00 h). The procedures in this study were per-
Ginkgo biloba and quercetin (Rojas et al. 2011; Holzmann formed in accordance with the National Institute of Health
et al. 2015), Curcuma longa and curcumin (Liao et al. 2013; Guide for the Care and Use of Laboratory Animals. The ex-
Xu et al. 2005a), and Hypericum perforatum and hyperforin periments were developed after the protocol approval by the
(Sharpley et al. 1998; Cervo et al. 2002). In addition, syringic Institutional Ethics Committee (CEUA/FURB – 007/15). All
acid (SA - 4-hydroxy-3, 5-dimethoxybenzoic acid) the major efforts occurred to minimize the animals’ suffering and to
benzoic acid and a phenolic compound derived from Morus reduce their numbers to the minimum necessary to demon-
nigra leaves have shown antioxidant (Memon et al. 2010), an- strate consistent effects in the experiments.
ticancer and hepatoprotective activities (Malhi et al. 2014).
Recently, SA also demonstrated antihyperglicemic property Drugs and treatments
(Srinivasan et al. 2014) and neuroprotective effect on mouse
model of Parkinson (Rekha et al. 2014). Morus nigra L. aqueous extract (MN, 3–100 mg/kg, p.o.),
Therefore, the goals of this study were to identify phenolics antidepressant fluoxetine (FL, 10 or 20 mg/kg, p.o., Cadila
from the aqueous extract of Morus nigra (MN) and investigate Healthcare, India) were dissolved in distilled water and
the antidepressant-like effect of MN, as well as, the involve- syringic acid (SA, 0.1–100 mg/kg, Sigma Chemical
ment of antioxidant and neuroprotective activities. Lastly, the Company, USA) was dissolved in saline containing 1% (v/v)
SA, the major phenolic constituent of M. nigra extract and the Tween 80. The administration was once (acute) or once a day
largely-known antidepressant, the fluoxetine, were also eval- in a period of 7 days (subchronic), both via oral route (p.o.) by
uated in the comparison. gavage, in a constant volume of 10 ml/kg body weight mice.
The tests (behavioral and biochemical) were performed
60 min or 24 h after the last administration, for the acute or
Material and methods subchronic treatment, respectively. Fluoxetine, used here as a
positive control, was administered at doses previously shown
Plant material and Morus nigra aqueous extract (MN) to cause antidepressant-like effects according to Lenzi et al.
preparation 2015. The dissolution of MN was freshly done from the ly-
ophilized power immediately before its administration by ga-
The Morus nigra L. leaves harvested on February, 2015, in the vage. Controls received an identical volume of saline contain-
city of Blumenau (Santa Catarina State, Southern Brazil - ing 1% (v/v) Tween 80.
Metab Brain Dis (2017) 32:1963–1973 1965

Behavioral tests Evaluation of lipid peroxidation

Forced swimming test (FST) Thiobarbituric acid reactive substances (TBARS) assay
levels determined according to the method described by
The FST was carried out individually in mice forced to swim Ohkawa et al. (1979) that measures malondialdehyde
in an open cylindrical container (diameter 10 cm, height (MDA), a product of lipoperoxidation caused mainly
25 cm), with 19 cm of water at 25 ± 1 °C. Each mouse was through hydroxyl free radicals read at 535 nm. The calibra-
made immobile, after it ceased struggling and remained float- tion curve developed using 1,1,3,3-tetramethoxypropane
ing motionless in the water, making only those movements and TBARS levels calculated as nanomol of
necessary to keep its head above water.The total immobility malondialdehyde formed per milligram of protein.
duration had been monitored during 6 min as described pre-
viously (Zeni et al. 2011). The observer was blind to the ani-
Protein carbonyl (PC) assay
mal treatments.
The protein carbonyl content was assayed following the meth-
Tail suspension test (TST) od of Reznick and Packer (1994), with modifications. After
the homogenate preparation, supernatant was discarded and
The total immobility duration induced by tail suspension mea- the pellet resuspended in 10 mM 2,4-dinitro-phenyl hydrazine
sured according to Steru et al. (1985). The animals were both (DNPH) in 2 M HCl and allowed to stand at room temperature
acoustically and visually isolated suspended 50 cm above the for 60 min, vortexing every 15 min. After protein precipita-
floor by adhesive tape placed approximately 1 cm from the tip tion, the pellet washed with 1 ml ethanol: ethyl acetate (1:1 v/
of the tail. The immobility time recorded during 6 min (Zeni v) solution, dissolved in 6 M guanidine hydrochloride and
et al. 2011). Mice were only considered immobile when hung centrifuged at 14000 rpm for 3 min. The samples were read
passively and completely motionless. The observer was blind against a complementary blank at 370 nm. A blank was run
to the animal treatments. with a parallel procedure with 2 M HCl alone. The carbonyl
content was expressed in nmol/mg of protein.

Open-field test
Nitrite determination
The behavior parameters assessed in the open-field test as
described previously by Lenzi et al. (2015). The open field The production of the nitrite levels determined based on
arena, a wooden box measuring 40 × 60 cm and 50 cm height Griess reaction (Griess 1879). After centrifugation (1800 g
with the floor divided into 12 equal squares. At the start of for 10 min) of the homogenate 50 μl of the supernatant was
each trial, a mouse was placed in the left corner of the field and incubated with 100 μl of Griess reagent at room temperature
freely allowed to explore the arena. Three observational mo- for 10 min and the absorbance was measured at 525 nm. The
ments were considered: the number of crossings (squares standard curve was prepared with several concentrations of
crossed with all paws) as indicative of locomotor activity, NaNO2 (ranging from 0 to 100 μM) and was expressed as
the number of rearings (the animal standing upright on its μmol/g of protein.
back legs) as exploratory behavior and the number of fecal
boluses as emotionality, registered during a period of 6 min. Determination of non-protein thiol groups (NPSH)
The arena floor was cleaned between the tests with a solution
of 10% ethanol and the tests carried out in a temperature, NPSH levels evaluated in order to estimate endogenous de-
noise, and light controlled room avoiding anxiety behavior. fenses against oxidative stress through the method based on
Ellman’s reagent (DTNB) reaction with free thiol groups.
Briefly, the samples were mixed with 0.4 M Tris–HCl buffer,
Biochemical analysis
pH 8.9 and 0.01 M DTNB, the NPSH level determined by the
absorbance at 405 nm and expressed as nmol of NPSH/mg of
After 60 min and 24 h to the last administration, in the acute
protein (Ellman 1959).
and subchronic treatments, respectively, mice were sacrificed
by decapitation. Immediately, the whole blood was collected
and serum separated by centrifugation (3000 x g, 10 mim) at Protein determination
room temperature. The homogenates prepared according to
Lenzi et al. (2015). At the same time, the whole brain were Protein measured by Lowry et al. (1951) method, using serum
removed from mice. bovine albumin as standard.
1966 Metab Brain Dis (2017) 32:1963–1973

Ex vivo neurochemical experiments Statistical analysis

Treatment of mice with MN and preparation of slices The results were expressed as means ± S.E.M or S.D. com-
parisons between treatments and control groups performed by
The MN (3, 10, 30 and 100 mg/kg) was administered once one-way analysis of variance (ANOVA) followed by Tukey’s
a day by gavage during seven days and 24 h after the last HSD test, when appropriate, using GraphPad Prism (version
treatment mice were killed by decapitation. Rapidly, the 5.0). A value of P < 0.05 was considered significant.
hippocampus and cerebral cortex removed and placed in
ice-cold Krebs–Ringer bicarbonate (KRB) buffer of the
following composition: 122 mM NaCl, 3 mM KCl,
1.2 mM MgSO 4 , 1.3 mM CaCl 2 , 0.4 mM KH 2 PO 4 , Results
25 mM NaHCO3 and 10 mM D-glucose. The buffer was
bubbled with 95% O 2 –5% CO 2 up to pH 7.4. Slices Phenolic analysis by high-performance liquid
(0.4 mm thick) were rapidly prepared using the McIlwain chromatographic (HPLC)
Tissue Chopper, separated in KRB at 4 °C and recovered
for 30 min in KRB at 37 °C. Hippocampal and cortical As shown in Fig. 1 the major compound identified in MN was
slices were incubated with glutamate (10 mM or syringic acid (80.57%). Effects of the treatments with Morus
100 mM, respectively - Sigma Chemical Company, USA) nigra aqueous extract (MN), syringic acid (SA) or fluoxetine
for 1 h in KRB. After this period, the medium was with- (FL) on the immobility time in the TST and FST and on the
drawn and replaced by a nutritive incubation medium com- locomotor, exploratory and emotionality activities in the
posed of 50% of KRB, 50% of Dulbecco’s modified open-field test in mice.
Eagle’s medium (DMEM, Sigma Chemical Company, The results depicted in Fig. 2a and b, show that the acute
USA), 20 mM HEPES and 100 μg/ml gentamicine, at treatment by oral route of MN,decreased the immobility time
37 °C in a CO2 atmosphere for additional 3 h in order to in the FST and TST, respectively, indicating the
evaluate cell viability (Zeni et al. 2011). antidepressant-like effect. SA treatment caused the same effect
(Fig. 2c). Statistical analyses revealed a significant effect of
MN treatment at all doses in the FST and TST (P < 0.001)
Evaluation of cell viability with the exception at the dose of 100 mg/kg, in the TST. Post
hoc analyses also indicated a significant decrease on the im-
Hippocampal or cortical cell viability was evaluated 3 h mobility time elicited by the SA treatment at the doses 1 and
after glutamate exposure. Cell viability was determined 10 (P < 0.001) but not at 0.1 or 100 mg/kg. Figure 2 shows
through the ability of cells to reduce MTT (3-(4,5-dimeth- that the fluoxetine treatment (10 or 20 mg/kg, p.o.) also pro-
ylthiazol-2-yl-diphenyltetrazolium bromide, Sigma duced a significant effect on the immobility time, in the TST
Chemical Company, USA) (Mosmann 1983). The slices or FST (P < 0.05 and P < 0.001, respectively). Figure 3 shows
were incubated with MTT (0.5 mg/ml) in KRB for 30 min that MN, FL and SA administered for 7 days also caused a
at 37 °C. The tetrazolium ring of MTT cleaved by active significant decrease on immobility time in the TST compared
dehydrogenases produce a precipitated formazan which to the control group (P < 0.001, P < 0.01 and P < 0.01, re-
was solubilized adding 200 μl DMSO, resulting in a col- spectively). As shown in Table 1, MN, FL or SA administered
ored compound whose optical density measured byan in acute or subchronic form did not change significantly the
ELISA reader (550 nm). number of crossings, rearings and fecal boluses in the open-

Fig. 1 Chromatographic profile

(HPLC) ethyl acetate fraction of
aqueous extract from Morus nigra
L. (MN). Identified peak
represent syringic acid
Metab Brain Dis (2017) 32:1963–1973 1967

As shown in Table 2, the statistical analyses revealed that

the acute treatment with MN caused a significant decrease of
TBARS level at 3 mg/kg dose (P < 0.001) in the serum and
therefore did not alter this parameter in the brain. Also, the FL
(10 mg/kg) acute treatment decreased lipid peroxidation in the
serum (P < 0.001) but increased it in the brain (P < 0.01),
while FL (20 mg/kg) did not alter TBARS level in the serum
or in the brain. Although, the MN subchronic treatment in-
creased the TBARS level in the serum (3, 10 and 100 mg/kg -
P < 0.001, P < 0.05 and P < 0.05) and in the brain (3 mg/kg -
P < 0.01), as well as, FL (10 mg/kg) rose the MDA measure-
ment in the serum (P < 0.001) and in the brain (P < 0.01).
Furthermore, the SA subchronic treatment demonstrated anti-
oxidant activity in the serum (P < 0.001) and pro-oxidant
effect in the brain (P < 0.001).
Also depicted in Table 2, the measurement of the nitrites
production evidenced that the MN did not decrease in acute
but in subchronic treatment diminished the nitrites level in the
serum (10–100 mg/kg - P < 0.001) and in the brain (30 and
100 mg/kg - P < 0.01 and P < 0.001, respectively). FL (10 and
20 mg/kg) acute or subchronically administered did not alter
the production of nitrites in the serum or in the mice’s brain.
Although, SA treatments were capable to counteract the pro-
duction of nitrites, not only in the serum (P < 0.001) but also
in the brain (P < 0.001).
The effects of the treatments with MN and SA on the PC
level are depicted in Fig. 4a and b, respectively. Only the dose
at 30 mg/kg (P < 0.05) was able to decrease significantly the
PC formation, as well as FL (P < 0.05) and SA (P < 0.001).
No significant changes were observed on the NPSH levels in
all the treatments tested (Fig. 4c and d).
Effect of the treatment with Morus nigra aqueous extract
(MN), syringic acid (SA) or fluoxetine (FL) against
glutamate-induced toxicity in the mice’s hippocampal and ce-
rebral cortex slices
The incubation of hippocampal and cerebral cortex
slices for 1 h with glutamate (10 and 100 mM, respective-
Fig. 2 Effect of the acute administration of Morus nigra aqueous extract ly) significantly reduced the cell viability when compared
(MN, 3–100 mg/kg, p.o.), fluoxetine (FL, 10 and 20 mg/kg, p.o.) or to control slices (P < 0.001). Glutamate-induced hippo-
syringic acid (SA, 0.1–100 mg/kg, p.o.) in the FST (panel a) and TST campal and cerebral cortex slices damages were not ob-
(panel b and c). Values are expressed as means ± S.E.M. (n = 5–8). *
served in hippocampal slices obtained from mice previ-
P < 0.05; ** P < 0.01 and *** P < 0.001 as compared with the vehicle-
treated control ously treated with MN, FL or SA in the ex vivo evalua-
tion of the neuroprotective effect (Figs. 5 and 6,
respectively). In fact, there was no toxicity observed
field test. These results indicated that their antidepressant-like through cell viability of hippocampal and cerebral cortex
effect was not either affected by the locomotor exploratory or slices. Furthermore, the MN (3–100 mg/kg, P < 0.001),
emotional activities. FL (10 mg/kg, P < 0.001) or SA (10 mg/kg, P < 0.001)
Effects of the treatments with Morus nigra aqueous extract subchronic treatment was able to counteract the
(MN), syringic acid (SA) or fluoxetine (FL) in the nitro- excitotoxicity damages in the hippocampus of mice (Fig.
oxidative stress parameters in the serum and the mice’s brain 5b and c). Moreover, MN (3–30 mg/kg, P < 0.001 and
Table 2 and Fig. 4 summarizes the effects of the treatments 100 mg/kg, P < 0.01) or SA (10 mg/kg, (P < 0.001) in the
on TBARS, nitrite, PC and NPSH levels, respectively, in the cerebral cortex of mice was significantly capable to
serum and mice’s brain. exert neuroprotection against glutamate (Fig. 6b and c).
1968 Metab Brain Dis (2017) 32:1963–1973

Fig. 3 Effect of the subchronic

administration of Morus nigra
aqueous extract (MN, 3–100 mg/
kg, p.o.), fluoxetine (FL, 10 mg/
kg, p.o.) or syringic acid (SA,
1 mg/kg, p.o.) in the TST (panel a
and b, respectively). Values are
expressed as means ± S.E.M.
(n = 6–8). ** P < 0.01 and ***
P < 0.001 as compared with the
vehicle-treated control

Discussion excitotoxicity evaluation revealed protection of the hippocam-

pal and cortical slices, demonstrating that MN is neuroprotec-
In the present study, we demonstrated for the first time that tive. Furthermore, we showed that SA, the major phenolic
acute and subchronic MN treatments have significant compound from MN could be responsible, at least in part, for
antidepressant-like effect with the involvement of antioxidant the effects exerted by MN since it presented the same effects.
activity in both tests, the FST and TST, without alterations on The FST and TST are widely used to screen newly antide-
the motor performance in mice. Additionally, glutamatergic pressant drugs, as they are sensitive to all major classes of

Table 1 Effect of Morus nigra

(MN), fluoxetine (FL) and Group/Dose mg/kg/day Treatments Parameters
syringic acid (SA) on number of
crossings, rearing and fecal Crossings Rearings Fecal boluses
boluses in the open field test in
mice Control Acute 142.0 ± 33.73 35.1 ± 9.53 1.0 ± 1.41
Subchronic 81.7 ± 37.19 18.2 ± 13.79 1.0 ± 1.06
MN 3 Acute 156.1 ± 23.55 34.1 ± 13.55 0.8 ± 1.05
Subchronic 93.5 ± 35.24 21.5 ± 9.51 1.7 ± 1.83
MN10 Acute 109.0 ± 42.40 32.0 ± 15.47 1.4 ± 1.51
Subchronic 93.0 ± 32.31 22.7 ± 17.89 1.0 ± 1.41
MN 30 Acute 87.8 ± 23.67 16.3 ± 15.04 1.3 ± 1.36
Subchronic 105.5 ± 33.18 31.0 ± 11.61 2.1 ± 1.64
MN 100 Acute 136.4 ± 58.96 39.8 ± 31.02 1.6 ± 2.19
Subchronic 114.0 ± 38.06 22.8 ± 17.56 1.7 ± 1.25
SA 0.1 Acute 116.8 ± 51.06 35.5 ± 15.45 0.8 ± 0.95
SA 1 Acute 96.5 ± 36.84 35.8 ± 7.88 1.0 ± 2.00
Subchronic 79.3 ± 47.99 34.4 ± 26.33 1.0 ± 1.22
SA 10 Acute 79.3 ± 47.99 39.0 ± 27.98 0.5 ± 0.57
SA 100 Acute 107.8 ± 48.47 37.0 ± 12.35 0.8 ± 0.95
FL 10 Acute 105.4 ± 23.10 27.8 ± 10.22 0.1 ± 0.35
Subchronic 93.8 ± 33.95 25.5 ± 17.30 0.5 ± 0.78
FL 20 Acute 90.7 ± 87.41 7.4 ± 6.22 0.3 ± 0.46

Mean ± DP. P > 0.05 (n = 5)

Metab Brain Dis (2017) 32:1963–1973 1969

Table 2 Effect of Morus nigra

(MN), fluoxetine (FL) and Group/Dose Treatments Parameters
syringic acid (SA) on TBARS and mg/kg/day
nitrite levels in the serum and in TBARS Nitrites
the mice’s brain
Serum Brain Serum Brain

Control Acute 1.73 ± 0.31 0.57 ± 0.01 0.15 ± 0.01 0.26 ± 0.05
Subchronic 1.55 ± 0.19 0.46 ± 0.03 0.17 ± 0.01 0.26 ± 0.01
MN 3 Acute 0.57 ± 0.12*** 0.73 ± 0.14 0.15 ± 0.02 0.25 ± 0.02
Subchronic 2.42 ± 0.26*** 0.77 ± 0.08** 0.19 ± 0.01 0.25 ± 0.03
MN 10 Acute 1.24 ± 0.24 0.54 ± 0.05 0.13 ± 0.01 0.28 ± 0.05
Subchronic 1.95 ± 0.20* 0.48 ± 0.03 0.11 ± 0.01*** 0.22 ± 0.01
MN 30 Acute 2.03 ± 0.17 0.43 ± 0.05 0.15 ± 0.02 0.24 ± 0.02
Subchronic 1.30 ± 0.08 0.51 ± 0.02 0.10 ± 0.01*** 0.19 ± 0.01**
MN 100 Acute 2.05 ± 0.47 0.68 ± 0.10 0.14 ± 0.02 0.26 ± 0.02
Subchronic 1.99 ± 0.30* 0.48 ± 0.01 0.12 ± 0.01*** 0.19 ± 0.01***
SA 1 Subchronic 0.84 ± 0.33*** 0.84 ± 0.07*** 0.04 ± 0.01*** 0.05 ± 0.01***
FL 10 Acute 0.52 ± 0.06*** 0.91 ± 0.11** 0.14 ± 0.01 0.26 ± 0.01
Subchronic 2.37 ± 0.20*** 0.82 ± 0.25*** 0.19 ± 0.01 0.27 ± 0.04
FL 20 Acute 1.67 ± 0.42 0.73 ± 0.05 0.12 ± 0.01 0.23 ± 0.01

Mean ± DP
*P < 0.05 significant difference as compared to control
**P < 0.01 significant difference as compared to control
***P < 0.001 significant difference as compared to control

antidepressant drugs (Porsolt et al. 1977; Steru et al. 1985) biphasic (‘bell-shaped’) dose–response curve in the TST.
with distinct neurochemical basis (Bai et al. 2001). Indeed, a biphasic dose–response has been reported in the
Therefore, the fact that MN administration exerted the effect TST or FST, with extracts, Aloysia gratissima (Zeni et al.
in both tests reinforces the notion that MN might play a role in 2011) and Valeriana glechomifolia (Müller et al. 2012), re-
the modulation of depression. However, MN (100 mg/kg) did spectively, or with compounds, 5,6-dibromo-N,N-dimethyl-
not alter de immobility time in the FST and SA caused a tryptamine and nortriptyline hydrochloride (Oliveira et al.

Fig. 4 Effect of the subchronic

administration of Morus nigra
aqueous extract (MN, 3–100 mg/
kg, p.o.), fluoxetine (FL, 10 mg/
kg, p.o.) or syringic acid (SA,
1 mg/kg, p.o.) on oxidative stress
damage in the brain. The effects
on protein carbonyl (PC) and
NPSH levels (panel a, b and c, d,
respectively). Values are
expressed as means ± S.E.M.
(n = 6–8). * P < 0.05; ** P < 0.01
and *** P < 0.001 as compared
with the vehicle-treated control
1970 Metab Brain Dis (2017) 32:1963–1973

Fig. 6 Ex vivo cell viability analysis in cerebral cortex slices incubated

Fig. 5 Ex vivo cell viability analysis in hippocampal slices incubated with glutamate (GLU, 100 mM) for 1 h. When present, Morus nigra
with glutamate (GLU, 10 mM) for 1 h. When present, Morus nigra aqueous extract (MN, 3–100 mg/kg, p.o.), Fluoxetine (FL, 10 mg/kg,
aqueous extract (MN, 3–100 mg/kg, p.o.), Fluoxetine (FL, 10 mg/kg, p.o.) or syringic acid (SA, 1 or 10 mg/kg, p.o.) was pre-administered in
p.o.) or SA (1 or 10 mg/kg, p.o.) was pre-administered in mice by gavage mice by gavage (7 days before slices preparation). After this period,
(7 days before slices preparation). After this period, incubation media was incubation media was withdrawn and replaced for fresh culture medium
withdrawn and replaced for fresh culture medium without GLU and without GLU and maintained for additional 3 h. Control group (first black
maintained for additional 3 h. Control group (first black bar) was consid- bar) was considered as 100% and represents cell viability of slices incu-
ered as 100% and represents cell viability of slices incubated only in bated only in culture medium. The values represent means ± SD of at least
culture medium. The values represent means ± SD of at least 5 experi- 5 experiments carried out in triplicates. * P < 0.05, **P < 0.01 and
ments carried out in triplicates. **P < 0.01 and *** P < 0.001 significant- *** P < 0.001 significantly different from control group (100%); ##
ly different from control group (100%); ### P < 0.001 significantly dif- P < 0.01 and ### P < 0.001 significantly different from GLU group
ferent from GLU group

the antidepressant-like effect in the TST. Since phenolic com-

1990; Diers et al. 2008). It has also been proposed that the pounds are well-known antioxidants and exerts antidepressant-
TST shows more sensitivity than FST to lower doses of anti- like effect, as resveratrol (Xu et al. 2010), curcumin (Xu et al.
depressants (Thierry et al. 1986). 2005a, b), fesitin (Zhen et al. 2012), quercetin (Holzmann et al.
In this study, MN and SA subchronically administered had 2015) and ferulic acid (Zeni et al. 2012), it is expected that the
repeated the results observed in the acute treatment confirming MN and SA influence the production of nitro-oxidative stress.
Metab Brain Dis (2017) 32:1963–1973 1971

In fact, we observed reduction of TBARS levels in the serum slices demonstrating the involvement of the neuroprotection
by MN or FL in the acute treatment and by SA in the in the antidepressant-like effect. Herewith, we introduced new
subchronic treatment. Although MN and FL in the repeated assumptions to show that MN and SA as potential
treatments increased the lipid peroxidation in the serum and antidepressive agents which may be further evaluated to im-
this also occurred in the brain of the animals treated with prove antidepressant therapy.
MN, SA or FL. Silva et al. (2010) demonstrated that ketamine,
10 and 20 mg/kg, acutely administered, decreased immobility
Acknowledgements This work was supported by grants from
time in the TST and increased TBARS level in the prefrontal Universidade Regional de Blumenau (FURB), PIBIC-FURB, PIBIC-
cortex of mice. We previously demonstrated a correlation be- CNPq and FAPESC scholarships. The authors thank the English language
tween decrease in TBARS level and antidepressant-like effect review by professors Luiz Henrique da Silva and Marta Helena Caetano
in the TST (Lenzi et al. 2015) in the hippocampus and in the (FURB). There is a patent request (PI10 2016 029764 8, INPI, Brazil)
associated with this study.
cerebral cortex of mice chronically treated with ferulic cid
showing a modulation of this system. Considering these find-
ings, herein we used the whole brain in the measurement of
TBARS level, which pointed out a limitation of a comparison. References
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