Effects of lemon balm (Melissa officinalis) on behavioral deficits and memory impairment of rats surviving sepsis

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Vol. 11(8), pp. 153-160, 25 February, 2017
DOI: 10.5897/JMPR2016.6266
Article Number: 9325A2663135
ISSN 1996-0875
Copyright © 2017
Author(s) retain the copyright of this article
http://www.academicjournals.org/JMPR
Journal of Medicinal Plants Research
Full Length Research Paper
Effects of lemon balm (Melissa officinalis) on
behavioral deficits and memory impairment of rats
surviving sepsis
João Eudes Filho1, Dâmaris Silveira1, Aluízio Carlos Soares1, Fabiana Pirani Carneiro1,
Melissa Sousa de Assis1, Franco Batista Leite1, Niraldo Paulino2, Greice Maria Souza1,
Mônica Valero da Silva1, Stéphanie Marchiori1, Karla Amaral1, Nadyelle Targino de Melo1,
Vania Moraes Ferreira1*
1Universidade de Brasília, Campus Universitário Darcy Ribeiro, s/n, 70910-900, Brasília, DF, Brazil.
2Medical Lex Gestão de Informações e Cursos Ltda. Av. Desembargador Vitor Lima, 260, sala 908, Ed. Madson Center
- Trindade, 88040-400, Florianópolis, SC, Brazil.
Received 2 October, 2016; Accepted 22 February, 2017
Sepsis has become one of the most frequent causes of mortality in intensive care centres. So far, there
is no effective pharmacotherapy that can prevent or improve the neurological consequences and
enhance survival. The goal of this study is to investigate the neuroprotective effect of lemon balm
(Melissa officinalis) on behavioural dysfunctions produced in sepsis-surviving rats. Adult male rats
were subjected to caecal ligation and puncture and the control animals were submitted to the sham
operation. Lemon balm ethanolic extract or saline, given orally, was administered for one week after
surgery procedures. Locomotion, anxiety, depressive behaviour and memory were investigated. In the
elevated plus-maze (EPM), the percentage of open arm entries and open arm time was very significant
in the animals treated with lemon balm extract, similar to the diazepam response in sham-operated and
sepsis-surviving rats. Locomotion in open field tests and the enclosed arm entries in the EPM were not
significantly altered by treatments. In the forced swimming (FS) test, the extract was effective at
reducing the immobility time as that demonstrated by fluoxetine. In the step-down inhibitory avoidance
test, the extract eased the effects on memory of sepsis-surviving animals. Collectively, these results
demonstrate that lemon balm ethanolic extract could be used for the prevention of cognitive and mood-
related deficits that may be associated with sepsis sequelae.
Key words: Anxiety, depression, locomotion, Melissa officinalis, memory.
INTRODUCTION
Brain disorders are currently getting top priority among
the science target and the literature has shown the
behavioural problems associated with traumatic brain
injury. Sepsis is an example of this damage and it is
characterized by a systemic inflammatory response and
is associated with high mortality in humans (Greenberg et
al., 2014). The main reason of death in septic patients
could be encephalopathy (Mazeraud et al., 2016; Tauber

154 J. Med. Plants Res.
et al., 2017). Nowadays, several studies are conducted to
evaluate the damage to the brain during sepsis and its
behavioural consequences, especially involving cognitive
and mood impairments present in sepsis survivors
(Comim et al., 2016; Leite et al., 2013; Mazeraud et al.,
2016; Tauber et al., 2017). The polymicrobial sepsis
induced in rats by caecal ligation and perforation (CLP)
experimental models are important because they
reproduce human sepsis consequences (Leite et al.,
2013), which is clinically relevant for elucidating new
therapy alternatives for this health problem (Hutchins et
al., 2014).
Traditional medicine was used on people before the
great development of modern medicine, based on
multiple alternatives of natural products to treat several
physiological dysfunctions. Research into the historical
literature has demonstrated that some of the natural
products and/or their pharmacologically active principles
have a broad spectrum of biological activities, including
central nervous system (CNS) properties (Andrade et al.,
2016; Bu et al., 2016).
Piper nigrum L. (Piperaceae) used in traditional
medicine of many countries, for example, has different
properties, such as antiinflammatory, antioxidant,
antidepressant and cognitive effects. The methanolic
extract of their fruits in beta-amyloid rat model of
Alzheimer's disease significantly exhibited anxiolytic- and
antidepressant-like effects and also antioxidant potential
(Hritcu et al., 2015). Acute treatment of rats with Lindera
obtusiloba extracts, commonly used as an alternative
medicine in Korea, significantly reduced immobility time
and increased swimming time, suggesting that the
antidepressant-like effects of this extracts were likely
mediated via the glucocorticoid receptor (Lim et al.,
2016). In addition, other neuroprotective efficacy could be
observed in in vivo and in vitro experiments (Jalsrai et al.,
2016).
Melissa officinalis is a medicinal plant from the
Lamiaceae family that has been used as a folk medicine
to threat central nervous disorders (Anheyer et al., 2017;
Kennedy et al., 2002). This plant has neuroprotective
activity at CNS because it displays potent antioxidant
activity (Soodi et al., 2017). From the phytochemical point
of view, the main chemical principles of this species of
herbal medicine are flavonoids, alkaloids, phenolic acids
and tannins (Noguchi-Shinohara et al., 2015; Shakeri et
al., 2016). These compounds may contribute to the
homeostasis of the CNS, especially because they can
protect against neurodegeneration and/or behavioural
disorders that can be correlated to dementia or traumatic
brain injury. Notably, it has also been reported as
lowering excitability, cognitive dysfunctions, stress and
anxiety levels in rodent models and humans (Anheyer et
al., 2017; Lin et al., 2015; Ross, 2015; Shakeri et al.,
2016). Previous results from our lab showed that the oral
intake of lemon balm EE for about 10 days has anxiolytic
and antidepressant‑like properties in a manner gender
(male vs. female) dependent, particularly with regard to
the effective dose for each exposure length (Taiwo et al.,
2012).
Despite great availability of information regarding the
neuroprotective effects of plant extracts, the options for
sepsis treatment is scarce. In this context, the goal of this
work is to verify the possible neuroprotective action of
lemon balm extract on behavioural and cognitive deficits
due to sepsis sequela in rats.
MATERIALS AND METHODS
Animals
Male Wistar rats (n= 60), 3 months old, 350 g, from the animal
facility of Sena Aires Faculty (Valparaíso de Goiás-GO, Brazil),
were housed 5 to a cage with food and water available ad libitum
and were maintained on a 12-h light/dark cycle (lights on at 7:00
am) at room temperature (23 ± 1°C). The animals were allowed to
adapt to the laboratory conditions for at least 1 week before the
behavioural assessment. All procedures used in the present study
complied with the guidelines on animal care of the UNB Ethics
Committee on the Use of Animals, which follows the “Principles of
laboratory animal care”. Subjects were transferred in their cages to
the experimental room on the day of being tested, where they were
left unbothered to habituate for at least 1 h before the behavioural
tests.
Preparation of the plant extract
Dried leaves of M. officinalis were kindly supplied by Centroflora
Group (Botucatu, Brazil), who also provided a certification of the
plant’s identity and quality. Powdered plant material (1900 g) was
extracted by maceration at room temperature (24±3°C) using
ethanol as solvent. Following removal of the solvent, under vacuum
conditions and below 40°C using a Heildolph system (Heildolph ®
Instruments, Germany) composed by a rotavapor coupled to a
vacuum pump D-91126, and chiller a MX07R-20HD2E. After
solvent elimination, the crude ethanolic extract (EE) that was
obtained (13% yield) was then stored at -18°C.
Drugs and pharmacological procedures
Lemon balm EE (100 mg/kg) was dissolved in a solution containing
150 µL of Tween 80, 150 µL of ethanol and 150 µL of
Dimethylsulfoxide (DMSO). All these substances (except EE) were
obtained from Sigma Aldrich, Brazil. Each resulting preparation was
then suspended in 0.9% physiological saline. An equivalent
*Corresponding author. E-mail: vmmf@unb.br. Tel: + 55 61 8122 0005.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License

preparation of Tween/alcohol/DMSO/saline (this mixture was used
to avoid fluid overload, swelling, acidosis and high blood sodium,
and to produce the best dilution of the drugs. Saline was used to
dilute other medications to be given by injection) was used as
vehicle control. These solutions were prepared 24 h prior to being
administered via oral gavage, in a volume of 1 mL/kg, having been
stored at 4C. Diazepam (1 mg/kg; DZP; Roche, Brazil) and
fluoxetine (10 mg/kg; FXT; Bluepharma, Brazil) were suspended in
distilled water containing 2% Tween 80 and physiological saline
(DMSO is not necessary in this solution, only for lemon balm EE
due to the product consistency), respectively. Both drugs were
prepared on the same day as being administered by gavage, in a
volume of 1 mL/kg. Lemon balm EE dose selection was based on
previous study published elsewhere (Kennedy et al., 2002). The
doses of the other substances varied according to the
pharmaceutical efficacy of each of them, in accordance with clinical
practice.
Caecal ligation and perforation surgery
Rats were weighed and anesthetized with a mixture of ketamine (80
mg/kg) and xylazine (10 mg/kg), given intraperitoneally. A 2-cm
midline laparotomy was performed to allow exposure of the caecum
to the adjoining intestine. The caecum was tightly ligated 5 times
with a 4.0-silk suture (Bioline, Brazil) at its base, below the
ileocaecal valve, and it was perforated five times through-and-
through with a 14-gauge needle (BD, Brazil). The caecum was then
gently squeezed to extrude a small amount of faeces from the
perforation site, returned to the peritoneal cavity, and the
laparotomy was closed with 4.0 silk sutures. The sham-operated
rats were submitted to all surgical procedures previously described
but the caecum was not perforated. To reproduce the protocol used
in surgery procedures on humans, all animals received saline
immediately after surgery procedures (50 mL/kg subcutaneous). A
mixture of ceftriaxone (30 mg/kg) + clindamycin (25 mg/kg) was
administered by intraperitoneal route (i.p.) every 12 h for 3 days.
They were kept in groups of 5 animals per cage with free access to
food and water, according to the procedure performed elsewhere
(Leite et al., 2013).
Treatment protocols
The animals were classified into 2 groups: the sham-operated
group and the sepsis group. Each one was divided into 3
subgroups to receive by oral route the control solution (saline);
diazepam (1 mg/kg) or fluoxetine (10 mg/kg), as positive controls;
or lemon balm EE (100 mg/kg). Animals received daily treatments
for 7 days and they were tested one hour after the seventh
administration. On the test day, one hour after treatment, the
animals were subjected to the behavioural tests. The doses chosen
in these protocols were based on the literature or previous
experiments from the laboratory.
Experimental procedures
Open field test
To evaluate locomotor activity, animals were tested in an open field
(OF), consisting of a wooden square box (60 x 60 x 35 cm) divided
into nine equal squares (20 x 20 cm). Each animal was placed in
the centre of the field and left to explore freely for 5 min. The
number of quadrants crossed with all four paws was used as a
measure of locomotion. After each trial, the apparatus was cleaned
Filho et al. 155
with ethanolic solution (10% v/v).
Elevated plus-maze test
Measures of anxiety were obtained in the elevated plus-maze
(EPM) test. This apparatus is made of wood, comprised of two
opposing open arms (50 x 10 cm) and two opposing closed arms
(50 x 10 x 40 cm), interconnected by a common central platform (10
x 10 cm). The entire apparatus was elevated 50 cm above floor
level, but to prevent falls, both open arms had a 1 cm high
plexiglass edge. The animal was placed on the central platform,
facing an enclosed arm, and allowed to explore the maze freely for
5 min. The following parameters were scored: number of open and
enclosed arm entries, time spent in the central platform, open and
enclosed arms. These were used to calculate the percentages of
open arm entries (%OAE) and open arm time (%OAT). Arm entry
was defined as all four paws in a specific arm (Pellow et al., 1985).
After each trial, the EPM was cleaned with ethanolic solution (10%
v/v).
Forced swimming test
The forced swimming (FS) test was used, modified from that
described by Porsolt et al. (1977). The animal was placed, for 5
min, in a glass cylinder (30 cm in diameter and 50 cm height)
containing 40 cm of water at 23±1°C, which forced the rat to swim
or float as its hind limbs were unable to touch the bottom. Time
spent immobile was recorded only during the last 3 min. Immobility
was scored whenever the animal stopped swimming and floated,
making only the small limb movements necessary to keep its head
above water level. After 5 min, the animal was removed from the
apparatus and dried.
Step-down inhibitory avoidance test
The inhibitory avoidance apparatus (Insight, Brazil) is to evaluate
aversive memory. It consisted of an acrylic box (50 × 25 × 25 cm),
whose floor was made of parallel stainless-steel bars (1 mm
diameter) spaced 1 cm apart. A platform (7 cm wide × 2.5 cm high)
was placed on the floor against the left wall. The animals were
placed on the platform and their latency in stepping down on the
grid with all four paws was measured with an automatic device. The
animals were submitted to the inhibitory avoidance task using a
protocol similar to that described previously (Lucena et al., 2013).
During training sessions, immediately after stepping down on the
grid, the animals received a 0.4-mA, 1.0-s (controlled by a monitor
attached to the equipment) scrambled foot shock. During test
sessions, no foot shock was administered and the step-down
latency (maximum 180 s) was used as the measure of retention.
The animals were submitted to a single training session.
In order to evaluate memory, test sessions were performed 1.5 h
after training. The administration of the extract or control solution
was performed by oral route 1 h before the training in the inhibitory
avoidance apparatus.
Statistical analysis
Data were expressed as means ± mean standard error (s.e.m.) of
10 animals and were analysed using one-way analysis of variance
(ANOVA) followed by Tukey's test. Data on the inhibitory avoidance
test are shown as median (interquartile range) of step-down
latencies. Comparisons of test session step-down latencies

156 J. Med. Plants Res.
Figure 1. Effects of oral subchronic administration of
saline (SAL), diazepam (DZP) and Melissa officinalis
ethanolic extract (100 mg/kg) on the percentage of open
arm entries (A), on the percentage of open time spent (B)
and on the number of enclosed arm entries (C) in the
elevated plus-maze test on sham-operated and sepsis-
surviving rats. Each bar represents the mean ± S.E.M. of
10 animals per group. *p≤0.05 as compared to the saline
treatment of the SHAM group; #p≤0.05 as compared to
the SEPSIS group (ANOVA, Tukey’s test).
between groups were performed with the Kruskal–Wallis test
followed by the Mann-Whitney test. Data were analysed by Graph
Pad Instat® (GraphPad software, San Diego, CA). P-values lower
than 0.05 (P<0.05) were considered statistically significant.
RESULTS
In the EPM, sham-operated rats that had been exposed
to lemon balm EE showed enhanced percentage of open
arm entries (Figure 1A): [F(5,59) = 5.00, p<0.05] and
percentage of open arm time (Figure 1B): [F(5.59) = 4.20,
p<0.05], similar to the animals treated with DZP: % open
arm entries (Figure 1A): [F(5.59) =5.06, p<0.05] and %
open arm time (Figure 1B): [F(5,59) = 4.91, p<0.05]. In the
sepsis-surviving rats, the extract also enhanced the
percentage of open arm entries (Figure 1A): [F(5.59) =
5.91, p<0.05] and the percentage of open arm time
(Figure 1B): [F(5.59) = 5.07, p<0.05], similar to the animals
treated with DZP: % open arm entries (Figure 1A): [F(5.59)
=5.96, p<0.05] and % open arm time (Figure 1B): [F(5.59) =
5.00, p<0.05]. The frequency of enclosed arm entries
(Figure 2C) in the EPM as well as the number of
quadrants crossed in the open field test (Figure 2) were
not affected by any treatment and remained constant
between groups.
As shown in Figure 3B, FXT treatment was able to
reduce the immobility time in sham-operated ([F(5.59) =
3.96, p<0.05]) and sepsis-surviving rats ([F(5.59) = 3.21,
p<0.05]) as compared to the group that received the
control solution in each group. Lemon balm EE in sepsis-
surviving rats modified the immobility time only in the
sepsis groups ([F(5.59) = 5.37, p<0.05]).
As shown in Figure 4, the Kruskal-Wallis test followed
by the Mann-Whitney test revealed that the lemon balm
EE administration was able to improve the memory-
facilitating effect produced by sepsis-surviving rats in the
model of the step-down inhibitory avoidance test when
measuring short- ([H(4, N=31) = 27.10, p<0.0001]) and
long-term memory -([H(4, N=31) = 24.80, p<0.0001]).
DISCUSSION
Lemon balm EE was effective in reducing anxiety,
depression and cognitive impairment levels produced by
sepsis in rodents, showing a property independent of any
sedative effect as demonstrated by no interference in
locomotion in the open field test and frequency of
enclosed arm entries in the EPM, as demonstrated by
Figures 1 to 4. Most of the time, the phytomedicine
showed a similar effect with the positive controls DZP
and FXT, suggesting that the interference in GABA and
5-HT receptors by this extract may be a new alternative
for treating neurological dysfunctions produced by sepsis.
Anxiety and depression are the most prevalent mental
disorders arising among child, adolescent and adult
patients. Both psychiatric illnesses demonstrate marked
overlap and co-occurrence (Freeman et al., 2002).
Despite this, the prevalence of anxiety is 15 to 25% and
the prevalence of depression appears to be higher than
50% (Brown et al., 2001; Lewinsohn et al., 1997). These
two disorders may share the same or different
vulnerability factors even though anxiety disorders may
represent causal risk factors for the development of
depression (Goes, 2015; Starr et al., 2014).
0
20
40
60
*
*
**
A##
% Open arm entries
0
10
20
30
40
*
*
**
B
##
% Open arm time
SAL
DZP
M100
SAL
DZP
M100
0
2
4
6
8
10
SHAM
C
SEPSIS
Enclosed arm entries
Open arm entries (%)
Open arm time (%)

Filho et al. 157
Figure 2. Effects of oral subchronic administration of saline (SAL), diazepam
(DZP) and M. officinalis ethanolic extract (100 mg/kg) on the number of
quadrants crossed in open field test in sham-operated and sepsis-surviving
rats. Each bar represents the mean ± S.E.M. of 10 animals per group.
Figure 3. Effects of oral subchronic administration of saline (SAL), fluoxetine
(FXT) and Melissa officinalis ethanolic extract (100 mg/kg) on the immobile
time (in seconds) evaluated in the forced swimming test in sham-operated and
sepsis-surviving rats. Each bar represents the mean ± S.E.M. of 10 animals
per group. *p≤0.05 as compared to the saline treatment of the SHAM group;
#p≤0.05 as compared to the FXT group (ANOVA, Tukey’s test).
In the context of actual research, it was noted that the
sepsis-surviving rats showed suggestive anxiety behaviour
that was observed by reduction in the percentage of open
arm entries and the time spent in those arms in the EPM,
characterizing anxiogenic response. Those rats receiving
lemon balm EE maintained a similar response to the
0
20
40
60
80
SAL DZP M100 SAL DZP M100
SHAM SEPSIS
Locomotion
SAL
FXT
M100
SAL
FXT
M100
0
20
40
60
80
SHAM SEPSIS
**
#
#
Immobility (sec)

158 J. Med. Plants Res.
Figure 4. Effects of oral subchronic administration of saline (SAL) and
Melissa officinalis ethanolic extract (100 mg/kg) on short- (A) and long-
term memory (B) in the step-down inhibitory avoidance test in sham-
operated and sepsis-surviving rats. Data are shown as the median
(interquartile ranges) of 10 animals. *p<0.05 as compared to the saline
treatment of the respective group (Kruskall Wallis - Mann Whitney).
sham animals. As regards the assessment of depression,
the animals showed increased immobility time in the
forced swimming test, which was reduced by FXT and
the lemon balm EE. These anxiolytic and antidepressant
results were not due to either hypoactivity or hyperactivity
displayed by animals, since their locomotion was not
changed in the open field test and the entries in the
enclosed arms in the EPM.
The observed anxiolytic effect of this natural product is
congruent with data obtained elsewhere (Kennedy et al.,
2006). The results are supported by the fact that rodents
normally exhibit a preference for the enclosed arms and
avoidance of the open arms of the EPM. Anxiolytic drugs,
such as DZP, modulate EPM behaviour in rodents,
causing an increase in the percentage of entries and time
spent in the open arms of the maze. Therefore, these
measures can serve as an index of anxiety (Horii and
Kawaguchi, 2015).
Thus, our results are in accordance with previous data,
showing anxiogenic and depressive responses in sepsis-
surviving rats. Leite et al. (2013) observed that repeated
nicotine administration does not alter the survival rate in
rats subjected to caecal ligation and puncture and they
provide new evidence that nicotine can improve long-
lasting memory impairment and anxiogenic-like responses
in sepsis-surviving animals. Tuon et al. (2007), in turn,
demonstrated in sepsis-surviving rats, a significant
increase in the immobility time as compared to the sham-
operated rats. The imipramine was able to reduce this
evaluated parameter, with no locomotion impairment. In
0
30
60
90
120
150
180
*
*
A
Latency (sec)
SAL
M100
SAL
M100
0
30
60
90
120
150
180
**
B
SHAM SEPSIS
Latency (sec)

the experiments, the FXT, a selective serotonin reuptake
inhibitor, showed similar results.
Lemon balm is known for its mild sedative properties
(Anheyer et al., 2017), even following a single
administration (Soulimani et al., 1991). In our study, such
behaviour was not observed. The fact that the lemon
balm EE and DZP did not generally alter quadrant
crossings and enclosed arm entry in the sham and EPM
tests, respectively, suggests that the psychoactive effects
observed in the present study may not be due to changes
in the animals’ locomotor patterns.
Regarding the cognitive aspect, animals that survived
the sepsis showed a loss of short- and long-term
memory, which was characterized by the decrease in
length of time spent in the inhibitory avoidance platform.
The administration of lemon balm EE made the animals
spend more time on the platform, characteristic behaviour
of memory retention.
Several studies have recently shown the importance of
phytotherapies for treating behavioural dysfunctions and
cognitive problems in humans (Anheyer et al., 2017;
Kennedy et al., 2002; Ross, 2015), even though there is
little information about the possible pharmacodynamics
involved in the pharmacological effects. For M. officinalis,
it is well documented that its effects could be related to
the active components that have been identified as
monoterpenoid aldehydes, monoterpene glycosides,
flavonoids and polyphenolic constituents (Shakeri et al.,
2016; Wightman, 2017).
Substantial progress has been made in our
understanding of how some herbal medicines can
ameliorate behavioural and cognitive processes in
patients with different neurological impairments. As
previously mentioned, nature has sources for various
types of treatment, providing effective antimicrobials to
treat infections caused by bacteria, viruses and fungi,
while offering therapeutic support to multiple organs and
body systems. On account of this, in this present
research, we also do not rule out the possibility that
lemons balm EE have interfered in aspects directly
related to inflammation, since it possesses potential anti-
inflammatory activities, supporting the traditional
application of this plant in treating various diseases
associated with inflammation and pain in more peripheral
areas (Bounihi et al., 2013) and brain areas, whose
active principles can probably be carried through the
cerebral blood flow and interact with several
neurotransmitters and signalling pathways involving
kinase enzymes (Wightman, 2017).
Conclusion
Although, there is a wide variety of a drug to treat
microbial infections, it is important to consider the
frequency and severity of adverse effects. Moreover, as
Filho et al. 159
the arsenal of available antibiotics in the pharmaceutical
market is increasingly being depleted due to microbial
resistance, there is a need for more alternative and less
toxic treatments. Thus, considering the results obtained
here, it is possible to suggest that the extract from lemon
balm can be considered a potential alternative for the
treatment of diseases, which have similar (or no similar)
reliability as conventional drugs DZP and FLX.
CONFLICTS OF INTERESTS
The authors have not declared any conflict of interests.
ACKNOWLEDGEMENTS
The authors thank MSc Elizabeth Tèran (in memorian)
from Centroflora (Ourinhos, Brazil) for generously
providing the Melissa officinalis extract used in this study.
They are also grateful to Sena Aires Faculty for providing
the experimental animals used in this study.
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