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Neuroprotective and Neurological Properties of Melissa officinalis

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Melissa officinalis has traditionally been used due to its effects on nervous system. Both methanolic and aqueous extracts were tested for protective effects on the PC12 cell line, free radical scavenging properties and neurological activities (inhibition of MAO-A and acetylcholinesterase enzymes and affinity to the GABA(A)-benzodiazepine receptor). The results suggest that the plant has a significant (P < 0.05) protective effect on hydrogen peroxide induced toxicity in PC12 cells. The radical scavenging properties were also investigated in cells and in cell free systems, where this plant was shown to be a good free radical scavenger. The MAO-A bioassay was also performed to detect possible antidepressant activities demonstrating that both extracts inhibited this enzyme, which has a key role in neurotransmitters metabolism. However, no activity was detected in the acetylcholinesterase and GABA assays. In general, the methanolic extract was more effective than the aqueous.
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ORIGINAL PAPER
Neuroprotective and Neurological Properties of Melissa officinalis
´ctor Lo
´pez ÆSara Martı
´nÆMaria Pilar Go
´mez-Serranillos Æ
Maria Emilia Carretero ÆAnna K. Ja
¨ger Æ
Maria Isabel Calvo
Accepted: 18 April 2009 / Published online: 7 May 2009
ÓSpringer Science+Business Media, LLC 2009
Abstract Melissa officinalis has traditionally been used
due to its effects on nervous system. Both methanolic and
aqueous extracts were tested for protective effects on the
PC12 cell line, free radical scavenging properties and
neurological activities (inhibition of MAO-A and acetyl-
cholinesterase enzymes and affinity to the GABA
A
-ben-
zodiazepine receptor). The results suggest that the plant has
a significant (P\0.05) protective effect on hydrogen
peroxide induced toxicity in PC12 cells. The radical
scavenging properties were also investigated in cells and in
cell free systems, where this plant was shown to be a good
free radical scavenger. The MAO-A bioassay was also
performed to detect possible antidepressant activities
demonstrating that both extracts inhibited this enzyme,
which has a key role in neurotransmitters metabolism.
However, no activity was detected in the acetylcholines-
terase and GABA assays. In general, the methanolic extract
was more effective than the aqueous.
Keywords Melissa officinalis Traditional medicine
Neuroprotective PC12 Antioxidant MAO
Introduction
The leaves of Melissa officinalis L. (Lamiaceae), also
known as lemon balm, are used in traditional medicine to
prepare a tea for its nerve calming and spasmolytic effects
[14] although there are a great variety of phytopharma-
ceutical preparations containing this plant or its extracts.
Furthermore, this plant is used by food industry to flavour
different products due to its particular taste.
The number of people suffering from neurological dis-
orders has lately increased worldwide, specially in the
developed countries [5,6]. Between them, neurodegener-
ative diseases (Parkinson, Alzheimer) as well as psychiatric
ones (anxiety and depression) are the most common [7].
Oxidative stress is directly implicated in neurodegenera-
tive diseases. Reactive oxygen species are a class of highly
reactive molecules derived from oxygen and generated by
metabolic processes and some external factors. An excessive
production of ROS may cause DNA mutation and protein
and lipid oxidation leading to cellular senescence and neu-
ronal death [8]. Natural antioxidants from plants are well
known to protect the human organism from free radicals,
preventing from certain diseases. In this sense, plants are a
source of compounds with antioxidant activity and Melissa
officinalis might have some protective effects.
Neuroprotection was studied using an in vitro cellular
model with the PC12 (rat pheochromocytoma) cell line,
which retains features of dopaminergic neurons and is a
common model used in neurosciences [9,10]. Antioxidant
capacities were carried out in cell free systems with dif-
ferent free radicals.
Neurological activities were investigated because lemon
balm is mainly used for its calming effects: monoamine
oxidase (MAO) and acetylcholinesterase (AChE) inhibition
and affinity to the GABA
A
-benzodiazepine receptor.
V. Lo
´pez (&)M. I. Calvo
Department of Pharmacy and Pharmaceutical Technology,
School of Pharmacy, University of Navarra, Irunlarrea sn,
31080 Pamplona, Spain
e-mail: vlopezra@alumni.unav.es
V. Lo
´pez S. Martı
´nM. P. Go
´mez-Serranillos
M. E. Carretero
Department of Pharmacology, School of Pharmacy,
Complutense University of Madrid, 28040 Madrid, Spain
V. Lo
´pez A. K. Ja
¨ger
Department of Medicinal Chemistry, Faculty of Pharmaceutical
Sciences, University of Copenhagen, Universitetsparken 2,
2100 Copenhagen O, Denmark
123
Neurochem Res (2009) 34:1955–1961
DOI 10.1007/s11064-009-9981-0
Depression is a common form of mental illness. The
WHO estimates that at least 3% of the world’s population is
suffering from depression [11]. The sympthoms of depres-
sion can be alleviated by increasing the synaptic availability
of monoamines. Such increase can be brought about by
decreasing the metabolism of these neurotransmitters by
inhibition of oxidative enzymes as for example MAO-A.
Alzheimer’s disease is the most common form of dementia
and the current treatments include acetylcholinesterase
inhibitors such as galantamine. Binding to the GABA-ben-
zodiazepine receptor was also studied because affinity to this
site produces a sedative and anticonvulsive effect.
Studies on neuroprotective and neurological activities of
this plant may demonstrate the effects of Melissa officinalis
on the central nervous system as well as to elucidate the
mechanisms involved in the activity.
Experimental Procedure
Chemicals
The cytotoxicity detection kit was purchased from Roche
(Indianapolis, USA). Dulbecco’s Modified Eagles Medium
(DMEM), penicillin–streptomycin, fetal bovine serum
(FBS), horse serum (HS), sodium pyruvate and Dulbecco’s
phosphate buffered saline (PBS) were obtained from Gibco
(Barcelona, Spain). 2,2-azino-bis(3-ethylbenzothiazoline-
6-sulfonic acid) diammonium salt (ABTS), xanthine, xan-
thine oxidase from buttermilk, nitroblue tetrazolium salt
chloride (NBT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT), acetylthiocholineiodide
(ATCI), 5,50-dithiobis[2-nitrobenzoic acid] (DNTB) and
some others were purchased from Sigma-Aldrich (Spain).
Caffeic acid was obtained from Extrasynthe
`se (Genay,
France).
3
H-Ro 15-1788 (flumazenil) was purchased from
Perkin Elmer Life Sciences.
Plant Material
The plant was collected in Pamplona (province of Navarra,
Spain) in June 2004 and was authenticated by Silvia
Akerreta and Rita Yolanda Cavero (Department of Plant
Biology, University of Navarra). Voucher specimens
(18687) have been deposited in the PAMP Herbarium of
the University of Navarra.
Preparation of Extracts
The powder of dried aerial parts of the plants (5 g) was
successively extracted three times with 200 ml of dichlo-
romethane, ethyl acetate, methanol and water after macer-
ation at 4°C for 24 h. Solvents were removed under vacuum
until dryness in a rotatory evaporator and the aqueous
extracts were lyophilized. The dry extracts were kept in
glass vials at -40°C prior to biological assays. Only the
methanolic and aqueous extracts were used in these assays.
Extracts were dissolved in PBS and filtered before use.
Protective Effect Against H
2
O
2
-induced
Toxicity in PC12 Cells
Cell Culture
Rat pheochromocytoma cells (PC12) were obtained from
the American Type Culture Collection. Cells were grown
in DMEM supplemented with 10% heat-inactivated horse
serum, 5% heat-inactivated fetal bovine serum, penicillin
(10 U/ml), streptomycin (10 lg/ml) and 0.2 mM sodium
pyruvate. Cultures were incubated in the presence of 5%
CO
2
at 37°C and 100% relative humidified atmosphere.
MTT Assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) is a yellow tetrazolium salt that is con-
verted into a purple compound (formazan) in viable cells
by mitochondrial enzymes [12]. PC12 cells were seeded in
96-multiwell plates at 2 910
4
cells/well. After 48 h, plant
extracts were added to the wells at different concentrations
and incubated for 24 h. Cells were treated with PBS con-
taining H
2
O
2
250 lM for 30 min. After 24 h, the medium
was removed and MTT (0.3 mg/ml final concentration in
the wells) was added for 1 h incubation at 37°C. Cell
survival (%) was measured in terms of absorbance at
550 nm in a microplate reader (Bio-Tek, USA) comparing
treated (hydrogen peroxide or plant extract plus hydrogen
peroxide) with untreated cells.
LDH Assay
The protective effect of the extracts in the PC12 cell line
was also studied by the determination of lactate dehydro-
genase (LDH) into the incubation medium using a com-
mercial kit from Roche. LDH is a cytosolic enzyme
released into the medium when the integrity of the cell
membrane deteriorates suffering from necrotic cell death
[13]. Cells were seeded in 96-multiwell plates at 2 910
3
cells/well and treated as in the MTT assay. The assay was
carried out following the instructions of the manufacturer.
Background interferences were deducted by calculating the
LDH activity of the medium. Spontaneous release of LDH
was also calculated and subtracted by measuring LDH
activity of untreated cells. Total intracellular LDH was
measured in cell lysates obtained by treatment with the
manufacturer lysis solution. Percentage of cell death (LDH
1956 Neurochem Res (2009) 34:1955–1961
123
release) was calculated comparing LDH activity of treat-
ments with total intracellular LDH.
Measurement of Intracellular Reactive Oxygen Species
Formation
This assay was performed as previously described with
some modifications [14]. Cells were seeded in 96-multiwell
plates at 2 910
4
cells/well. After 48 h, cells were incu-
bated with 20,70-dichlorofluorescein diacetate (DCFH/DA)
for 30 min in a final concentration of 10 lM. DCFH-DA
solution was removed and cells were co-incubated with
different extract doses and 250 lMH
2
O
2
. The fluorescence
intensity was measured at 485 nm excitation and 530 nm
emission for 2 h.
Antioxidant Activity in Cell Free Systems
ABTS Assay
The free radical scavenging activity was measured as
previously described [15] but adapted to 96-well micro-
plates. About 200 ll of the ABTS solution was added to
20 ll of plant extracts at different concentrations. Absor-
bance was read after 6 min at 734 nm and percentage of
inhibition was calculated with the following equation:
(Abs
control
-Ab
sample
/Abs
control
)9100. The radical scave-
ging capacity (RSC) of the extracts is expressed as IC
50
,
defined as the concentration of extract that scavenge 50%
of free radicals. IC
50
values were calculated by a non-linear
regression with a one phase exponential association equa-
tion using GraphPad Prism version 4.0.
Superoxide Radical Scavenging Activity
Superoxide radicals were generated by the xanthine/xan-
thine oxidase (X/XO) system following a described pro-
cedure [16] with some modifications. The reaction mixture
contained 75 ll NBT (50 lM), 75 ll xanthine (145 lM),
75 ll plant extract (different concentrations) and 75 ll
xanthine oxidase (0.29 U/ml). The reaction was initiated
by the addition of the enzyme and the mixture was incu-
bated for 2 min at 37°C. Antioxidant activity was deter-
mined by monitoring the effect of the extracts on
the reduction of NBT to the blue chromogen formazan by
O
2-
at 560 nm: % RSC =(Abs
control
-Ab
sample
/Abs
control
)
9100. IC
50
values were calculated by a non-linear
regression using GraphPad Prism version 4.0.
Inhibition on Xanthine Oxidase
The effect of the extracts on xantine oxidase was also
evaluated at the same concentrations by measuring the
formation of uric acid from xanthine in order to detect
possible enzyme inhibition [17]. The reaction mixture
contained the same proportion of components as described
above except NBT: 75 ll phosphate buffer, 75 ll xanthine
(145 lM), 75 ll plant extract (different concentrations)
and 75 ll xanthine oxidase (0.29 U/ml). Absorbance was
read at 295 nm in this case after 2 min.
Neurological Activities
MAO-A Inhibitory Activity
The bioassay was performed in a 96-well microplate [18].
Each well contained fifty microliters plant extract (or
DMSO as blank), 50 ll chromogenic solution (0.8 mM
vanillic acid, 417 mM 4-aminoantipyrine and 4 U/ml
horseradish peroxidase in potassium phosphate buffer pH
7.6), 100 ll 3 mM tyramine and 50 ll 8 U/ml MAO-A.
Absobance was read at 490 nm every 5 min for 30 min.
Background interferences were deducted as the same way
described above but without MAO enzyme. Data were
analyzed using GraFit 5 to obtain IC
50
. Clorgyline was
used as a reference MAO-A inhibitor.
Acetylcholinesterase Inhibitory Activity
The activity was measured using a 96-microplate reader
based on Ellman’s method [19]. Each well contained 25 ll
of 15 mM ATCI in Millipore water, 125 llof3mM
DTNB in buffer C (50 mM Tris–HCl, pH 8, 0.1 M NaCl,
0.02 M MgCl
2
6H
2
O), 50 ll buffer B (50 mM Tris–HCl,
pH 8, 0.1% Bovine Serum), 25 ll plant extract in buffer A
(50 mM Tris–HCl, pH 8). The absorbance was read five
times every 13 s for five times at 405 nm. Then, 25 ll
0.22 U/ml AChE were added and the absorbance was
measured again eight times every 13 s at 405 nm. Galan-
tamine was used as control substance.
GABA
A
-receptor Assay
Membrane Preparation The preparation was performed
using cerebral cortex of four rats as described by Risa et al.
[20].
Flumazenil Binding Assay The membrane preparation
was washed with ice-cold buffer (50 mM Tris–citrate
pH 7.1). The suspension was centrifuged at 0–4°C for
10 min at 27,000g. The pellet was resuspended in Tris–
citrate buffer (2 mg original tissue per ml) and used for the
assay. 25 llof
3
H-Ro 15-1788 (flumazenil) was added to
25 ll of test solutions (1, 0.1, 0.01 mg/ml) and 500 llof
membrane preparation. Total and unspecific binding was
measured using buffer or diazepam (1 lM assay final
Neurochem Res (2009) 34:1955–1961 1957
123
concentration). After incubation for 40 min in an ice bath,
5 ml of ice-cold buffer was added to the samples and
poured onto Adventic glass fibre filters (GC-50) under
vacuum, and inmediatly washed with another 5 ml of ice-
cold buffer. The amount of radioactivity of the filters was
measured by conventional liquid scintillation counting
using Ultimo Gold XR as scintillation fluid. Clonazepam
was used as positive control.
Statistical Analysis
Data are means ±SD of at least three independent
experiments. One-way ANOVA followed by Dunnett’s
multiple comparison test was used to compare control and
treated groups in cells experiments. IC
50
values were
analyzed by t-test.
Results
Effect Against H
2
O
2
-induced Toxicity in PC12 Cells
The effect of the extracts in PC12 cells was determined
using the MTT, LDH and intracellular ROS formation
assays. Pretreatments with the methanolic extract at con-
centrations of 60 and 80 lg/ml protected PC12 cells
against hydrogen peroxide toxicity by the MTT assay.
Neuroprotection was also observed by the LDH test at the
same doses (Tables 1,2). Aqueous extract obtained from
Melissa officinalis did not protect cells by either MTT or
LDH assays.
For the study of the preventive effect on ROS formation,
cells were co-incubated with extracts and 250 lMH
2
O
2
.
Both aqueous and methanolic extracts reduced the per-
centage of ROS formation for the 2 h of the experiment
(Figs. 1,2).
Antioxidant Activity in Cell Free Systems
Methanolic and aqueous extracts possessed high antioxi-
dant properties in the cell free system experimental models.
Table 1 Protective effect of Melissa officinalis extracts on hydrogen
peroxide induced toxicity in PC12 cells by the MTT assay
Methanolic
extract
Aqueous
extract
Control (250 lMH
2
O
2
) 50.2 ±3.8 50.2 ±3.8
Treated (Melisa officinalis ?H
2
O
2
)
20 lg/ml 43.8 ±5.1 42.1 ±3.9
40 lg/ml 51.5 ±4.6 44.6 ±2.5
60 lg/ml 61.4 ±4.0* 50.4 ±6.1
80 lg/ml 64.4 ±7.3* 49.0 ±5.2
Results are expressed as % of cell survival (mean ±SD) of three
experiments performed in eight replicates. Concentrations 20–80 lg/ml
refer to the dose of extracts in the wells
*P\0.05 versus control (250 lMH
2
O
2
)
Table 2 Protective effect of Melissa officinalis extracts on hydrogen
peroxide induced toxicity in PC12 cells by the LDH release assay
Methanolic
extract
Aqueous
extract
Control (250 lMH
2
O
2
) 56.0 ±5.1 56.0 ±5.1
Treated (Melisa officinalis ?H
2
O
2
)
20 lg/ml 54.7 ±6.8 61.9 ±6.1
40 lg/ml 41.9 ±6.2 47.6 ±3.2
60 lg/ml 38.6 ±6.4* 42.7 ±4.8
80 lg/ml 29.7 ±9.8* 62.7 ±6.7
Results are expressed as % of cell death in terms of LDH release
(mean ±SD) of three experiments performed in triplicates. Con-
centrations 20–80 lg/ml refer to the dose of extracts in the wells
*P\0.05 versus control (250 lMH
2
O
2
)
60
80
100
120
140
160
180
200
01530456090120
Time (min)
% ROS
Hydrogen Peroxide
250µM
20 µg/ml
40 µg/ml
60 µg/ml
80 µg/ml
100 µg/ml
120 µg/ml
60
80
100
120
140
160
180
200
01530456090120
Time (min)
% ROS
60
80
100
120
140
160
180
200
01530456090120
Time (min)
% ROS
Hydrogen Peroxide
250µM
20 µg/ml
40 µg/ml
60 µg/ml
80 µg/ml
100 µg/ml
120 µg/ml
Fig. 2 Protective effect of aqueous extract of M. offficinalis on ROS
intracellular production after exposition to 250 lMH
2
O
2
. Results are
means of three experiments, each one performed in eight replicates.
Error bars not shown as SD was less than 10%
60
80
100
120
140
160
180
200
01530456090120
Time (min)
% ROS
Hydrogen Peroxide
250µM
20 µg/ml
40 µg/ml
60 µg/ml
80 µg/ml
100 µg/ml
120 µg/ml
60
80
100
120
140
160
180
200
01530456090120
Time (min)
% ROS
60
80
100
120
140
160
180
200
01530456090120
Time (min)
% ROS
Hydrogen Peroxide
250µM
20 µg/ml
40 µg/ml
60 µg/ml
80 µg/ml
100 µg/ml
120 µg/ml
Fig. 1 Protective effect of methanolic extract of M. officinalis on
ROS intracellular production after exposition to 250 lMH
2
O
2
.
Results are means of three experiments, each one performed in eight
replicates. Error bars not shown as SD was less than 10%
1958 Neurochem Res (2009) 34:1955–1961
123
However, this activity was not superior to the control
substances used in the assays (Table 3).
Neurological Activities
Results on the MAO-A/AChE inhibition and binding the
GABA
A
-benzodiazepine receptor are shown on Table 4.
Both the methanolic and aqueous extracts inhibited MAO-A,
the methanolic extract being the most effective (Fig. 3)
whereas inhibition of AChE was not detected. Clorgyline
was used a a selective MAO-A inhibitor, resulting twenty
times more potent than the methanolic extract. Neither
methanolic nor aqueous extracts exerted affinity to the
GABA
A
receptor as flumazenil was not displaced from
binding the receptor.
Discussion
Oxidative stress produces cell death and reactive oxygen
species, including hydrogen peroxide, is involved in neu-
rotoxic events related to some neurodegenerative processes
such as Alzheimer’s disease. It is known than lemon balm
extracts contain phenolic compounds such as flavonoids
and phenolic acids [21] that may scavenge free radicals.
The antioxidant activity of those extracts have previously
been established in different experimental models [2125]
but the protective effects in a PC12 cell line have never
been reported. PC12 cells are a useful model in neurosci-
ence due to its phenotypic characteristics with sympathetic
neurons. Therefore, a protective activity in these cells may
mean that lemon balm might have a protective effect in
neurons. In this study, exposition of cells to 250 lMH
2
O
2
reduced cell survival around 50% and pretreatments with
methanolic extracts increased the survival in 15%. The
co-treatment of PC12 cells with extract (methanolic and
aqueous) and 250 lMH
2
O
2
produced a significant reduc-
tion in ROS intracellular formation. Therefore these results
suggest that Melissa officinalis exert protective activities in
the PC12 cell line and might protect neurons from oxida-
tive stress.
Both extracts exhibited a dose dependent antioxidant
activity in the cell free systems. In all cases, the methanolic
extract was more effective than the aqueous, with a lower
IC
50
. Three different methodologies were performed to
evaluate the antioxidant activity. The ABTS method is fast
and easy but the ABTS radical is not involved in human
biology; another method using the superoxide radical,
which is a physiological radical, was then performed. The
superoxide radical was generated by the X/XO system [26]
and both extracts showed superoxide radical scavenging
activities. Taking into account that the scavenging effect on
superoxide radical might be due to an inhibition of the
enzyme, the effect of the extracts on XO was checked out.
The results show that only the methanolic extract of
Melissa officinalis had a weak inhibition on XO, with a
IC
50
value around 500 lg/ml, a hundred times lower effect
than the xanthine oxidase inhibitor allopurinol. The aque-
ous extract acted as a superoxide scavenger but no inhi-
bition on XO was detected. The results on antioxidant
activity were in part expected as this species is known to
Table 3 Antioxidant activity of Melissa officinalis in cell free
systems
IC
50
(lg/ml)
ABTS Superoxide
radical
Xanthine
oxidase
Methanolic extract 12.7 ±0.4** 5.9 ±0.9* 500.2 ±47.1
Aqueous extract 17.1 ±0.9 7.8 ±0.8 n.d.
Caffeic acid 2.2 ±0.4 1.4 ±0.2
Allopurinol 5.5 ±0.3
Results are presented as IC
50
values (mean ±SD) obtained from non
linear regression of three experiments performed in triplicates. IC
50
is
defined as the concentration of extract that inhibits 50% of the free
radical or the enzyme
*P\0.05; ** P\0.01 versus aqueous extract
Table 4 Neurological activities of Melissa officinalis
MAO-A AChE assay GABA
A
assay (% Flumazenil binding)
IC
50
(lg/ml) IC
50
(lg/ml) 10 mg/ml 1 mg/ml 0.1 mg/ml
Metanolic extract 19.3 ±2.3** na 78.7 ±2.3 94.7 ±16.1 84.7 ±16.1
Aqueous extract 48.2 ±5.8 na 94.8 ±9.0 94.3 ±4.4 95.2 ±11.3
Clorgyline 0.1 ±0.01
Galantamine 19.9 ±4.8
Results expressed as IC
50
for MAO-A and AChE assays. Results for GABA
A
assay are expressed as % of flumazenil binding (IC
50
for
clonazepam is 0.002 nm). In all cases, data (mean ±SD) were obtained from three experiments performed in triplicates
na no activity
** P\0.01 versus aqueous extract
Neurochem Res (2009) 34:1955–1961 1959
123
contain caffeic acid derivatives and flavonoids, which have
previously shown free radical scavenging properties [27].
As far as neurological activities are concerned only
inhibition of MAO-A was detected. It is the first time this
activity is reported for Melissa officinalis. MAO-A is an
enzyme distributed in the central nervous system and has a
key role in catecholamine metabolism. The inhibition of
this enzyme is related to alleviation of depression symp-
toms [7] so this could be one of the mechanisms involved
in its central nervous system effects as in many cases the
antidepressant activity is related to an anxyolitic effect.
Though lemon balm is usually used for its calming effect,
methanolic and aqueous extracts did not bind to the
GABA
A
receptor as previous studies demonstrated [28].
On the contary, Melissa officinalis from Lebanon showed
some kind of activity in the GABA-receptor assay [29].
Some previous reports also show the lack of activity of the
plant in the acetylcholinesterase assay [30]. Despite of the
unclear results about the methanolic and aqueous extracts
in the GABA assays, some authors found the affinity to the
GABA-receptor in the essential oil obtained from lemon
balm [31], which may be in major part, the responsible for
the anxyolitic effect.
Lemon balm contains some flavonoids such as quercitrin
as well as apigenin and luteolin derivatives that might
inhibit monoamine oxidases as previous studies demon-
strated that a number of flavonoids possesed MAO-inhib-
itory activity [3234].
In conclusion, Melissa officinalis has shown protective
effects in the PC12 cell line, free radical scavenging
properties and MAO-A inhibitory effects, the methanolic
extract being the most effective. These results suggest the
potential use of this plant or its constituents for central
nervous system disorders and as a neuroprotective agent to
prevent diseases in which oxidative stress is involved.
Acknowledegments University of Navarra Foundation and Alumni
Navarrensis Association are thanked for finantial support and
fellowships.
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[Aqueous extract ]
10-3 10-2
% MAO-A inhibition
0
20
40
60
80
[Methanolic extract]
10-3 10-2
% MAO-A inhibition
0
20
40
60
80
Fig. 3 MAO-A inhibitory effect of lemon balm extracts. Extracts were assayed at concentrations of 3.75, 7.5, 15, 30 and 60 lg/ml. Data are
means ±SD of three independent experiments performed in triplicates
1960 Neurochem Res (2009) 34:1955–1961
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... Although, analysis show that GABA and acetylhydrolyase have no biological action. Generally, the methanolic extract with methanol proved to be efficacious than compared with extract of water (López et al., 2009). ...
Chapter
Extensive research suggests that a number of plant-derived chemicals and traditional Oriental herbal remedies possess cognition-enhancing properties. Widely used current treatments for dementia include extracts of Ginkgo biloba and several alkaloidal, and therefore toxic, plant-derived cholinergic agents. Several non-toxic, European herbal species have pan-cultural traditions as treatments for cognitive deficits, including those associated with aging. Acute administration has also been found to reliably improve mnemonic performance in healthy young and elderly cohorts, whilst a chronic regime has been shown to attenuate cognitive declines in sufferers from Alzheimer's disease. The present chapter looks at the ethnobotanical and pharmacological importance of various plants cognitive enhancing and other neuroprotective abilities.
... The extracts also showed good free radical scavenging activity. Both extracts inhibited MAO-A, but no activity was detected on the acetylcholinesterase and GABA [82]. ...
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Neuroprotection is the preservation of the structure and function of neurons from insults from cellular injuries caused by a variety of agents or neurodegenerative diseases. Medicinal plants possess neuroprotective effects via mechanisms that include inhibiting protein-based deposit accumulation, oxidative stress, and neuroinflammation, and correcting defects of neurotransmitters such as acetylcholine and dopamine [1-3]. The current review will highlight the neuroprotective effects of medicinal plants.
... Lemon balm has been widely used in Asian traditional medicine to treat many psychiatric disorders, such as depression, anxiety, insomnia, anxiety-induced palpitation, and stress (Shakeri et al., 2016). In clinical studies on the CNS system, lemon balm has shown anxiolytic, anti-depressant, anti-insomnia, and neuroprotective effects (Lopez et al., 2009), and its effects on improving mood, cognitive performance, and memory function have been investigated (Shakeri et al., 2016;Ulbricht et al., 2005). ...
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A systematic review and a meta-analytic approach were considered to investigate the effects of lemon balm as a medicinal herb on anxiety and depression in clinical trials and its side effects. All randomized clinical trials published up to October 30, 2020 that examined lemon balm in patients with symptoms of depression or anxiety , with acute or chronic manifestations, were searched in 12 online databases. Statistical analysis was performed using RevMan software. Continuous data were analyzed using standardized mean differences. Statistical heterogeneity was assessed using Chi 2 , I 2 , and p value tests. Based on meta-analysis results, lemon balm significantly improved mean anxiety and depression scores compared with the placebo (SMD:-0.98; 95% CI: À1.63 to À0.33; p = 0.003), (SMD:-0.47; 95% CI: À0.73 to À0.21; p = 0.0005) respectively, without serious side effects. Current evidence suggests that lemon balm may be effective in improving anxiety and depressive symptoms , particularly in the acute setting. Due to the high level of heterogeneity between studies, results should be interpreted with caution. The small number of clinical trials and differences between their methods were the limitations of the present study. Further high-quality studies are needed to firmly establish the clinical efficacy of the lemon balm. K E Y W O R D S anxiety, depression, lemon balm, Melissa officinalis, systematic review
... Previous studies have shown that, compared to healthy people, the calm of healthy people is significantly improved, in addition to improving mood and cognitive abilities. 13,14 Some studies also support the effect of lemon balm in reducing agitation in patients with dementia. 15 "The presumed biologically active compounds in lemon balm include monoterpenic aldehydes, flavonoids, and polyphenolic compounds, such as rosmarinic acid and monoterpenic glycosides." ...
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Background: This study was pointed to evaluate the efficacy and safety of valerian and lemon balm additional to the quetiapine in critically ill patients with delirium and agitation. Methods: We conducted a randomized, double-blind, placebo-controlled study. Fifty-three adult intensive care unit (ICU) patients (according to ICU Confusion Assessment Method scores) who were treated for delirium received quetiapine and Neurogol syrup (a combination of valerian and lemon balm) or placebo 5 mL every 12 hours for five consecutive days. Improvement in agitation according to the Richmond Sedation and Restlessness Scale was considered the main outcome. Results: The trial was completed for 53 patients (27 in the treatment group and 26 in the placebo group). The baseline characteristics between the groups were similar. In the treatment group, the number of agitated patients was significantly reduced and the difference was statistically significant (p = 0.000). Compared with the placebo group, the length of ICU stay in the treatment group was significantly reduced (p = 0.001). The Glasgow Coma Scale improved significantly at the end of day 5 (p = 0.04). There was no statistical difference in the improvement of delirium between the study groups (p = 0.14). Neurogol syrup was well tolerated. Conclusion: The addition of Neurogol to quetiapine (a combination of valerian and lemon balm) can reduce agitation and shorten the length of stay in the ICU without adverse effects. Clearly, more research is still needed to investigate the role of herbal medicines in ICUs and their efficacy and safety. How to cite this article: Alikiaie B, Shahmoradi E, Yekdaneh A, Mousavi S. Addition of Valerian and Lemon Balm Extract to Quetiapine Reduces Agitation in Critically Ill Patients with Delirium: A Pilot Randomized Clinical Trial. Indian J Crit Care Med 2021;25(7):785-790.
... Researches have confirmed that the mechanism of action in some herbal medicines, such as lavender, with mood elevating effects, may be modulation of neurological pathways (GABAergic systems) [22].Previous researches have shown that the methanol extract of Mellisa officinalis L. represented MAO-inhibitory activity because of its flavonoids [23]. The antidepressant-like effects of M. officinalis leaves ethanol extract has been also reported in the forced swim test [24]. ...
... Tables 2 and 3 in alphabetical order, along with their scientific name plant part, solvent extract, percentage concentration, and inhibition zone of enzyme. For acetylcholinesterase (AChE) inhibitory activity, three Corydalis species have been tested while, in Danish folk medicine, methanol and aqueous extracts of 11 plants are used for improvement of cognition and memory [65]. ...
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Alzheimer's disease (AD) is the most prevalent form of dementia. Improving the amount of acetylcholine in the brain is an efficient way to treat the illness. The global incidence of dementia is estimated to be as high as 50 million, and it is expected to increase every 20 years until 2040, resulting in a costly burden of disease. Early-life risk factors for pathology include genes, chromosomal abnormalities, head injury, insulin resistance, and inflammation. Potentially modifiable risk factors including obesity, diabetes, hypertension, and smoking are associated with Alzheimer's disease (AD) and represent promising targets for intervention. The drugs currently being used to manage AD have various drawbacks. The chemical inhibition of cholinesterase enzymes is an effective technique for treating signal related neuropathology, and possible sources of compounds with these properties are natural products and biogenic metal oxide nanoparticles. There is a potential source of AChE and BChE inhibitors in the abundance of plants in nature, and natural goods appear to offer useful medications and templates for the development of other compounds. This dissertation represents a review of the literature on species of medicinal plants and nanomaterial related plants tested for their inhibitory action of AChE and BChE. Plant species and the plant-mediated metal oxide nanoparticles referred to are possible cholinesterase inhibitors and can assist researchers in their study of natural products that may be beneficial in the treatment of AD.
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In the treatment of tobacco use disorder, current approaches focus on pharmacotherapy, nicotine replacement, and psychotherapy. However, traditional treatments have been widely used in societies for the purpose of smoking cessation for years. Although cases using traditional herbs in the self-treatment of addiction have been reported in the literature, studies on this subject are very limited. Research on certain herbs shows that they may be effective in the treatment of tobacco use disorder by different mechanisms, however, there is no evidence that they are safe to consume as cigarettes. This article aims to question the place of traditional herbs in tobacco use disorder treatment through a case who started to smoke Melissa officinalis herb to help his nicotine withdrawal.
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Consideration and improvement for anxiety and depression are important during global pandemic diseases. Appropriate healthcare can be obtained by paying more attention to traditional medicinal sciences. The adverse effects of stress with its various symptoms can be managed by introducing plants that boost mental health. The most relevant psychological reactions in the general population related to global pandemic are pervasice anxiety, frustration and boredom, aspecific and uncontrolled fear, disabling loneliness, significant lifestyle changes, and psychiatric conditions. Ginseng, chamomile, passionflower, herbal tea, lavender, saffron, kava, rose, cardamom, Chinese date and some chief formula like yokukansan, Dan-zhi-xiao-yao-san, so-ochim-tang-gamiband, and saikokaryukotsuboreito are notable herbal treatments for mental health problems. The most common medicinal plants which have been used in Iran for the cure of stress and anxiety are Viper,s-buglosses, Dracocephalum, valerian, chamomile, common hop, Hawhorns, and Lavender. Medicinal plants and herbs can be used for treatment and alleviating negative effects of stress, anger and depression during the global pandemic.
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Anecdotal evidence from Danish folk medicine suggests that the tea of Calluna vulgaris Hull. has a nerve calming effect [1]. Unpublished results from our department show that the methanol extract of C. vulgaris has an inhibitory effect on monoamine oxidase A (MAO-A). For bioassay-guided isolation, dried, ground aerial parts of C. vulgaris (100g) were defatted twice with 1000ml heptane. The plant material was extracted twice with 1000ml methanol. The combined methanol extracts were evaporated to dryness, re-dissolved in 1000ml 80:20 methanol: water and partitioned three times against 300ml heptane. Methanol was removed from the methanol: water fraction by evaporation and the volume was made up with water. The resulting water fraction was partitioned three times against 300ml ethyl acetate. The three fractions (heptane, ethyl acetate and water) were tested for MAO-A inhibitory activity [2]. The active ethyl acetate fraction was fractionated on a VLC column (240g silica gel 60), eluted with heptane, mixures of heptane: ethyl acetate: methanol, mixtures of dichloromethane: methanol and methanol. A total of 34 fractions were spotted on a TLC plate and fractions showing similar patterns were combined. The fractions were tested in the MAO-A assay. The most active fraction was investigated by HPLC (C18) employing a gradient from 0 to 100% acetonitrile over 30min, revealing one major peak in the chromatogram. This peak was active in the MAO-A assay. The compound was re-crystallised from acetone yielding 14.3mg pure compound which was investigated by ¹H-NMR. By comparison with ¹H-NMR literature data [3, 4] the compound was identified as quercetin. LC-MS analysis revealed a molecular weight of 302.1g/mol confirming the identification. The IC50-value of quercetin was 18±2µM in the MAO-A assay (clorgylin: 0.2±0.02µM). References: 1. Brøndegaard, V.J. (1978) Folk og flora. Rosenkilde og Bagger. Denmark. 2. Stafford, G.I. et al. (2007) S. Afr. J. Bot. 73:384–390. 3. Moon, J. et al. (2001) Free Radic. Biol. Med 11:1274–1285. 4. Dutta, N.K. et al. (2007)J. Med. Pharm. Chem. 41:37–39.
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A method for the screening of antioxidant activity is reported as a decolorization assay applicable to both lipophilic and hydrophilic antioxidants, including flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants. The pre-formed radical monocation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS*+) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants. The influences of both the concentration of antioxidant and duration of reaction on the inhibition of the radical cation absorption are taken into account when determining the antioxidant activity. This assay clearly improves the original TEAC assay (the ferryl myoglobin/ABTS assay) for the determination of antioxidant activity in a number of ways. First, the chemistry involves the direct generation of the ABTS radical monocation with no involvement of an intermediary radical. Second, it is a decolorization assay; thus the radical cation is pre-formed prior to addition of antioxidant test systems, rather than the generation of the radical taking place continually in the presence of the antioxidant. Hence the results obtained with the improved system may not always be directly comparable with those obtained using the original TEAC assay. Third, it is applicable to both aqueous and lipophilic systems.
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New studies have implicated both the amyloid-precursor gene on chromosome 21 and the apolipoprotein-E (APOE) gene on chromosome 19 in the etiology of subtypes of Alzheimer's disease (AD). Clinical trials have demonstrated that tacrine, a cholinesterase inhibitor, has real, but limited use as a treatment for AD. Genetic testing of individuals at risk for Huntington's disease (HD) is posing new challenges for therapy and genetic counseling.
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Oxidative stress (OS) has been implicated in various degenerative diseases in aging. In an attempt to quantify OS in a cell model, we examined OS induced by incubating for 30 min with various free radical generators in PC12 cells by using the dichlorofluorescein (DCF) assay, modified for use by a fluorescent microplate reader. The nonfluorescent fluorescin derivatives (dichlorofluorescin, DCFH), after being oxidized by various oxidants, will become DCF and emit fluorescence. By quantifying the fluorescence, we were able to quantify the OS. Our results indicated that the fluorescence varied linearly with increasing concentrations (between 0.1 and 1 mM) of H2O2 and 2,2′-azobios(2-amidinopropane) dihydrochloride (AAPH; a peroxyl radical generator). By contrast, the fluorescence varied as a nonlinear response to increasing concentrations of 3-morpholinosydnonimine hydrochloride (SIN-1; a peroxynitrite generator), sodium nitroprusside (SNP; a nitric oxide generator), and dopamine. Dopamine had a biphasic effect; it decreased the DCF fluorescence, thus acting as an antioxidant, at concentrations