Fitoterapia 91 (2013) 261271

Contents lists available at ScienceDirect

Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Rosmarinus ofcinalis L. leaf extract improves memory

impairment and affects acetylcholinesterase and
butyrylcholinesterase activities in rat brain
Marcin Ozarowski a,b,, Przemyslaw L. Mikolajczak a,c, Anna Bogacz a,e, Agnieszka Gryszczynska a,
Malgorzata Kujawska d, Jadwiga Jodynis-Liebert d, Anna Piasecka g, Hanna Napieczynska c,
Micha Szulc c, Radoslaw Kujawski a, Joanna Bartkowiak-Wieczorek a,e, Joanna Cichocka a,
Teresa Bobkiewicz-Kozlowska c, Boguslaw Czerny a,f, Przemyslaw M. Mrozikiewicz a,e
a

Institute of Natural Fibres and Medicinal Plants, Wojska Polskiego 71b, 60-630 Poznan, Poland
Department of Pharmaceutical Botany and Plant Biotechnology, Poznan University of Medical Sciences, Sw. Marii Magdaleny 14, 61-861 Poznan, Poland
Department of Pharmacology, Poznan University of Medical Sciences, Rokietnicka 5a, 60-806 Poznan, Poland
d
Department of Toxicology, Poznan University of Medical Sciences, Dojazd 30, 60-631 Poznan, Poland
e
Laboratory of Experimental Pharmacogenetics, Department of Clinical Pharmacy and Biopharmacy, Poznan University of Medical Sciences, Swiecickiego 6, 61-781
Poznan, Poland
f
Department of General Pharmacology and Pharmacoeconomics, Pomeranian Medical University, Zolnierska 48, 70-204 Szczecin, Poland
g
Department of Pathogen Genetics and Plant Resistance, Metabolomics Team, Institute of Plant Genetics of the Polish Academy of Science, Strzeszynska 34, 60-479
Poznan, Poland
b
c

a r t i c l e

i n f o

Article history:
Received 1 May 2013
Accepted in revised form 20 September 2013
Accepted 22 September 2013
Available online 27 September 2013
Chemical compounds studied in this article:
Rosmarinic acid (PubChem CID: 5281792)
Huperzine A (PubChem CID: 1253)
Scopolamine hydrobromide (CID: 5184)
Carnosic acid (PubChem CID: 65126)
Carnosol (PubChem CID: 2579)

a b s t r a c t
Rosmarinus officinalis L. leaf as part of a diet and medication can be a valuable proposal for the
prevention and treatment of dementia. The aim of the study was to assess the effects of
subchronic (28-fold) administration of a plant extract (RE) (200 mg/kg, p.o.) on behavioral
and cognitive responses of rats linked with acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity and their mRNA expression level in the hippocampus and
frontal cortex. The passive avoidance test results showed that RE improved long-term memory
in scopolamine-induced rats. The extract inhibited the AChE activity and showed a stimulatory
effect on BuChE in both parts of rat brain. Moreover, RE produced a lower mRNA BuChE
expression in the cortex and simultaneously an increase in the hippocampus. The study
suggests that RE led to improved long-term memory in rats, which can be partially explained
by its inhibition of AChE activity in rat brain.
2013 Elsevier B.V. All rights reserved.

Keywords:
Rosemary
Memory
Acetylcholinesterase
Butyrylcholinesterase
Rat

Corresponding author at: Institute of Natural Fibres and Medicinal Plants, Wojska Polskiego 71b, 60-630 Poznan, Poland. Tel.: +48 61 6559550; fax: +48 61 6559551.
E-mail addresses: mozarow@ump.edu.pl (M. Ozarowski), przemmik@ump.edu.pl (P.L. Mikolajczak), aniabogacz23@o2.pl (A. Bogacz),
agnieszka.gryszczynska@iwnirz.pl (A. Gryszczynska), kujawska@ump.edu.pl (M. Kujawska), liebert@ump.edu.pl (J. Jodynis-Liebert), akar@igr.poznan.pl
(A. Piasecka), hanna.napieczynska@gmail.com (H. Napieczynska), mszulc@ump.edu.pl (M. Szulc), kujawskiradoslaw@gmail.com (R. Kujawski),
joanna@wieczorek.net.pl (J. Bartkowiak-Wieczorek), joanna.cichocka@iwnirz.pl (J. Cichocka), tbobkiew@ump.edu.pl (T. Bobkiewicz-Kozlowska), bczerny@wp.pl
(B. Czerny), pmm@post.pl (P.M. Mrozikiewicz).
0367-326X/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.tote.2013.09.012

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M. Ozarowski et al. / Fitoterapia 91 (2013) 261271

1. Introduction
Alzheimer's disease (AD) is a pathologically complex
disease implicating interactions between environmental and
genetic risk factors. Despite considerable advances in the
knowledge about Alzheimer's disease its etiology is still
based on the amyloid, the Tau and the cholinergic hypotheses
explaining the molecular mechanisms of AD [1,2].
Currently, it is believed that prolongation of the availability of
acetylcholine released into the neuronal synaptic cleft can
improve the cholinergic function in Alzheimer's disease by
inhibiting acetylcholine hydrolysis [3]. The cholinergic hypothesis of Alzheimer's disease proposes that the degeneration of
cholinergic neurons occurs in the basal forebrain and is
associated with the loss of cholinergic neurotransmission in
the cerebral cortex. This is therapeutically important since the
basal forebrain cholinergic system is known to be involved in the
cognitive processing of memory and attention [1]. The human
brain contains two major forms of cholinesterases: acetylcholinesterase (AChE, EC 2.3.1.6) and butyrylcholinesterase (BuChE,
EC 3.1.1.8). In the human brain, both AChE and BuChE are found
not only in neurons but also in astrocytes and oligodendrocytes,
as well as in neuritic plaques and tangles in AD patients, wherein
more recent studies have shown that the AChE is localized
mainly in the neurons, and BuChE is associated primarily with
glial cells as well as endothelial cells and neurons [4]. More
importantly, the AChE activity was shown to be reduced in the
cortex while the BuChE activity remains unchanged or increased
during AD development [5].
Some researchers claim that the use of nonselective
cholinesterase inhibitors which inhibit both BuChE and
AChE may be more beneficial to patients with Alzheimer's
disease. Moreover, both AChE and BuChE may be involved in
the pathology of the amyloid- peptide [5].
There have been long-established research trends into new
neuroprotective drugs from natural sources, which raise new
therapeutic hopes. One of the most promising medicinal plants
known for its antioxidant and neuropharmacological activity is
rosemary (Rosmarinus officinalis L.) of the Lamiaceae family
an aromatic, evergreen, shrubby herb widely distributed in the
Mediterranean region and also cultivated in Poland. Extracts of
rosemary leaves (RE) contain several fractions of biologically
active compounds, including essential oil, high percentages of
phenolic acids (e.g. rosmarinic acid (RA), chlorogenic acid),
phenolic diterpenes (e.g. carnosic acid, carnosol), pentacyclic
triterpenes (e.g. ursolic, oleanolic, betulic acid) and flavonoids
(e.g. derivatives of apigenin and luteolin) [69].
2. Aims
The main aim of this study was to assess the influence of
subchronic (28-fold) administration of RE on behavioral and
cognitive activities of rats and to evaluate the cholinesterase
(acetylcholinesterase and butyrylcholinesterase) activity and
gene expression level in the hippocampus and frontal cortex.
3. Materials and methods
3.1. Plant material
The leaves of R. officinalis L. (Lamiaceae) were obtained from
an herbal company Kawon-Hurt (Gostyn Wlkp., Poland) in

June, 2009. The plant material was identified by Prof. Jaromir

Budzianowski, Department of Pharmaceutical Botany and Plant
Biotechnology, Faculty of Pharmacy, Poznan University of
Medical Sciences. The voucher specimen (no. 15.173) has
been deposited in the Herbarium of the Institute of Natural
Fibres and Medicinal Plants in Poznan (Plewiska), Poland.
3.2. Preparation of the extract
1000 g of raw plant material was extracted with 50% ethanol
by percolation (24 h) at room temperature (22 1 C). After
filtration, the extract was concentrated under vacuum to
eliminate the ethanol content. The concentrated extract was
frozen and freeze-dried. The final product yielded 67.2 g of solid
extract.
3.3. Metabolite identication with HPLC-UV-MS
Identification of secondary metabolites present in the
R. officinalis leaves was performed using the HPLC/MS
system consisting of Agilent 1100 HPLC instrument with a
photodiode-array detector PDAe (Palo Alto, CA, USA)
and Esquire 3000 ion trap mass spectrometer (Bruker
Daltonics, Bremen, Germany) with the XBridge C18 column
(150 2.1 mm, 3.5 m particle size) and the MSn spectra
were recorded in the negative and positive ion modes using the
previously published approach [10,11]. The individual compounds were identified by comparison of mass spectra and
retention times to these of standard compounds and literature
data [7,9,1214]. The elution was conducted with water
containing 0.1% formic acid (solvent A) and acetonitrile
(solvent B). The gradient elution was started at 10% of B and
linearly changed to 20% of B in 10 min, then to 50% of B in
30 min and to 95% of B over 10 min, followed by the return to
stationary conditions and re-equilibration for 10 min.
All calculations were performed by external standardization by measurement of the peak areas.
3.4. Determination of total phenolic compounds in the extract
The calculation of polyphenols to gallic acid was done
using the FolinCiocalteu reagent with the spectrophotometric method described in European Pharmacopoeia 5th edition
(EurPh. 5.0).
3.5. Determination of total hydroxycinnamic acid derivatives
Determination of total hydroxycinnamic acid (HCA) derivatives calculated on RA was performed according to the
procedure in EurPh. 5.0.
3.6. Distillation of essential oil
The essential oil contents were determined by way of
stream distillation in a Deryng's apparatus according to EurPh.
5.0. 30.0 g of the leaves of R. officinalis (separate sample) was
placed in a round-bottom flask. 500.0 mL distilled water and
0.3 mL xylen were added and boiled in the Deryng's apparatus
for 3 h. The volume of the obtained essential oil was measured
in accordance with EurPh. 5.0 and used for chromatographic

M. Ozarowski et al. / Fitoterapia 91 (2013) 261271

analysis. 0.7 mL oil was obtained, i.e. 2.3% of total essential oil
contents.
3.7. Gas chromatography analysis
Gas chromatography (GC) analyses were carried out using a
Perkin-Elmer Clarus 500 gas chromatograph with a data
processing system and an FID (GC-FID). Separation was
achieved by using an Elite FFAP fused-silica capillary column
(30 m long, 0.32 mm in internal diameter, 0.25 m of film
thickness). The injector and detector temperatures were
220 C. Helium was used as a carrier gas with a flow of
1.5 mL min1. A sample of 1.0 L was injected, using slit mode
(split ratio 1:100). The results were reported as the relative
percentage of the total peak area.
3.8. Chemicals and drugs
All reagents for HPLC analysis, scopolamine hydrobromide
trihydrate (S) and reagents for biochemical analyses were
purchased from SigmaAldrich (Poland). Other substances
used in HPLC such as carnosol, carnosic acid, rosmarinic acid
were obtained from LGC Standard (Poland) and SigmaAldrich
(Poland). Huperzine A was obtained from Enzo Life Sciences AG
(Alexis Corporation, Biomibo Distribution, Poland). Chemicals
for gene expression analysis were obtained from Roche
Diagnostic and ALAB (Poland). All chemicals and drugs were ex
tempore prepared on the day of the experiment.

263

3.11. Cognitive and behavioral tests

3.11.1. Measurement of locomotor activity
Locomotor activity assessment was performed with a
licensed activity meter (Activity Cage, Ugo Basile, Italy) by
placing the animals in the centre of the apparatus and recording
their horizontal and vertical activity. The data obtained were
expressed as signals corresponding to animal movements for
5 min. The locomotor activity was measured 30 min after the
administration of a single dose of scopolamine or the vehicle
(H2O). Any distracting factors were reduced to the minimum
(noise, presence of people, presence of other rats).
3.11.2. Measurement of motor coordination
Motor coordination was evaluated using the chimney
test originally designed for mice [15]. Thirty min after
scopolamine or vehicle injection a rat was allowed to enter
a glass laboratory cylinder, 500 mm long and 80 mm in
diameter, placed on its side. Upon reaching the cylinder
bottom by the animal the position of the cylinder was rapidly
changed from horizontal to vertical and a timer was set off.
The rat began to move backwards immediately. The timer
was stopped after the rat left the cylinder and assumed a
sitting posture on the top of the vessel. The time of exit from
the cylinder was accepted as a measure of motor coordination. Motor impairment was assessed as the inability of rats
to climb backwards up the tube within 60 s. The test was
performed after 30 min following the administration of a
single dose of scopolamine or the vehicle.

3.9. Animals
Experiments with rats were performed in accordance
with Polish governmental regulations (Dz. U. 05.33.289). The
study was conducted in accordance with ethical research
guidelines on conscious animals, and the study protocol was
approved by the Local Ethics Committee on the Use of
Laboratory Animals in Poznan, Poland (64/2008).
The experiments were performed on male six week-old
Wistar rats housed in controlled room temperature (20 0.2
C) and humidity (6575%) under a 12 h: 12 h lightdark cycle
(lights on 7 a.m.). The animals were kept in groups of 810
each in light plastic cages (60 40 40 cm) and had free
access to standard laboratory diet (Labofeed B pellets) and tap
water.
3.10. Treatments
The waterethanol (1:1) extract from the leaves of
R. officinalis L. (RE) was administered intragastrically (p.o.) in a
dose of 200 mg/kg b.w. (groups RE + H2O and RE + S),
rosmarinic acid (SigmaAldrich) (RA) in a dose of 10 mg/kg
b.w. (p.o.) (groups RA + H2O and RA + S) and huperzine A in a
dose 0.5 mg/kg b.w. (p.o.) (groups huperzine A + H2O and
huperzine A + S) for 28 consecutive days. On the last day,
30 min after the last dose of RE, RA or huperzine A, scopolamine
(S) was given intraperitoneally (i.p.) in a dose of 0.5 mg/kg b.w.
Control groups were treated with 0.5% methylcellulose (MC),
and water for injection (H2O) was used as a vehicle for S (groups
MC + H2O and MC + S). Both RE and RA were prepared
ex tempore before administration and suspended in MC in
concentrations of 20 mg/mL and 10 mg/mL, respectively.

3.11.3. Measurement of cognitive activity

Cognitive activity was evaluated with the passive avoidance test and object recognition test.
3.11.3.1. Passive avoidance test. The passive avoidance test
was performed with a licensed apparatus (Passive Avoidance
System step-through, Ugo Basile, Italy) as a model for long
term memory assessment in animals (effects on retrieval and
memory consolidation) [16,17]. The test relies on rats'
natural preference of darkness. After 2 min of habituation to
the dark compartment a rat was placed in the illuminated
compartment and allowed to enter the dark compartment.
Two more approach trials were allowed on the following day
with a two minute interval between them. At the end of the
second trial, an unavoidable scrambled electric footshock
(500 A, AC, 3 s) was delivered through the grid floor of the
dark compartment (learning trial). Retention of the passive
avoidance response (latency) was tested 24 h later by
placing the animal in the illuminated compartment and
measuring the latency in re-entering the dark compartment
against the arbitrary maximum time of 180 s. The test was
performed after 30 min following the administration of a
single dose of scopolamine or the vehicle.
3.11.3.2. Object recognition test. The object recognition test
was used as a model for short term memory assessment in
animals [18]. The object recognition task took place in a
40 60-cm open box surrounded by 40-cm high walls made
of plywood with a frontal glass wall. All animals were
submitted to a habituation session during which they were
allowed to freely explore the open area for 5 min. No objects

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M. Ozarowski et al. / Fitoterapia 91 (2013) 261271

were placed in the box during the habituation trial. On the

day of testing, the animals were given an additional 3 min
re-habituation period prior to commencing the test. The test
was divided into three phases with two trials: acquisition (the
animals explored two identical objects (biologically inert
substance plastic, sufficient weight and were secured to the
floor of the arena) (A1 and A2) for a period of 3 min), inter-trial
interval (the animals were returned to the home cage for
30 min), retention (the animals explored a familiar object (A*)
that is a duplicate of those objects from the acquisition trial (to
minimize olfactory cues) and a novel object (B) for a further
3 min). Object exploration is defined by animals licking,
sniffing or touching the object whilst sniffing but not leaning
against, turning round, standing or sitting on the object. The
exploration times (s) of all objects were recorded with a
stopwatch for subsequent statistical analysis. The time measured as an exploration behavior was used to calculate a
memory discrimination index (OR): OR = (B A*)/(B + A),
where B was the time spent exploring the new object and A*
was the time spent exploring the familiar object. A higher OR
was considered to reflect a greater memory ability [18]. The
test was performed 30 min following the administration of a
single dose of scopolamine or the vehicle.
3.12. Acetylcholinesterase and butyrylcholinesterase activities
assay in rat brain
On the last day of the experiments, 60 min after the last
dose of RE, RA or huperzine A, the animals were killed by
decapitation, and the hippocampus and frontal cortex were
collected from the brains of the rats. The tissue samples were
then stored at 80 C until measurement of activity of
cholinesterases or their mRNA gene expression.
The activities of acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE) were performed by modified
Ellman's spectrophotometric method according to Isomae et al.
[19]. The activity of AChE and BuChE was determined by
measuring the formation of the yellow anions obtained from the
reaction between Ellman's reagent and the thiocholine generated by the enzymatic hydrolysis of acetylthiocholine iodide
(ATCh) and butyrylthiocholine (BTCh), respectively (sample
0.1 mL, PBS 0.8 mL, DTNB 0.1 mL, ATCh 0.20 mL and BTCh
0.20 mL). The biochemical assay of AChE and BuChE in the
homogenate of brain samples was expressed as mol/min/mg
protein by using spectrophotometric method (UV 412 nm).
3.13. RNA isolation and reverse transcription reaction
Total RNA isolation from the rat brain tissue homogenates
(frontal cortex, hippocampus) was carried out using TriPure
Isolation Reagent (Roche) according to manufacturer's protocol.
The integrity of RNA was visually assessed by conventional
agarose gel electrophoresis, and the concentration was evaluated by measuring the absorbance at 260 and 280 nm in a
spectrophotometer (BioPhotometer Eppendorf). RNA samples
were stored at 80 C until use. 1 g of total RNA from all
samples was used for the reverse-transcribed into cDNA using
the Transcriptor First Strand Sythesis Kit (Roche) according to
manufacturer's protocol. The obtained cDNA samples were
stored at 20 C or used directly for the quantitative real-time
PCR (qRT-PCR) reaction.

3.14. Real-time PCR mRNA quantication

The acetylcholinesterase (AChE), butyrylcholinesterase
(BChE), alpha- and beta-secretases genes expression levels
were analyzed by quantitative real-time PCR (RT-PCR) reaction
using a LightCycler TM Instrument (Roche, Germany) and a
LightCycler Fast Start DNA Master SYBR Green I kit (Roche
Applied Science), according to manufacturer's instructions. All
primer sequences were designed, and custom designed using
the Oligo 6.0 software (National Biosciences) and were verified
by assessment of a single PCR product on agarose gel and by a
single temperature dissociation peak (melting curve analysis) of
each cDNA amplification product. The GAPDH gene was used as
a housekeeping gene (endogenous internal standard) for
normalization of qPCR reaction. The relative quantification for
any given gene was expressed as a signal relative to the average
signal value for the internal standard. RT-PCR was carried out in
a reaction volume of 10 L reaction mixture with proper
concentrations of all components, according to manufacturer's
instructions. For each quantified gene, standard curves were
prepared from a cDNA dilution, and generated from a minimum
of four data points. Complementary DNA was quantified by
comparison of the number of cycles required for amplification of
unknown samples with those of the series of cDNA standard
dilutions. All quantitative PCR reactions were repeated twice.
The data were evaluated using the LightCycler Run 4.5 software
package (Roche Applied Science). Each PCR run included a
non-template control to detect potential contamination of
reagents.
3.15. Statistical analysis
All values were expressed as means SEM. The statistical
comparison of results was carried out using one-way analysis
of variance (ANOVA) followed by Duncan's post-hoc test for
detailed data analysis. The level of statistical significance was
set at p b 0.05.
4. Results
4.1. Cognitive and behavioral experiments
4.1.1. Locomotor activity
A one-way ANOVA analysis revealed significant differences
in the locomotor activity of rats expressed as their horizontal
spontaneous activity (F(7, 91) = 4.42; p b 0.001; Table 1).
Detailed post-hoc analysis showed that all substances
(RE + H2O, RA + H2O, huperzine + H2O and MC + S) did
not change the locomotor activity of rats. Huperzine A produced
a slight increase of this activity but the difference between the
obtained values and those by the control (MC + H2O) were
statistically non-significant (p b 0.1). Therefore, it can be stated
that the repeated administration of both RE and the substances
used in the experiment (huperzine A and RA) did not affect the
locomotor activity of rats (Table 1).
Different effects in the locomotor activity of rats were
observed after an acute S injection (Table 1). The S-treated
animals were found to produce significant differences in their
horizontal spontaneous activity (ANOVA: F(4, 65) = 4.81;
p b 0.01). The stimulation effect was produced by a single S
administration, but the detailed post-hoc analysis revealed a

M. Ozarowski et al. / Fitoterapia 91 (2013) 261271

265

(RA + S group) led to a statistically significant enhancement

of vertical activity when compared with the MC + S group
(p b 0.05), therefore RA stimulation also contributed to the
obtained total variability of the rats' vertical activity.

non-significant difference between S-treated animals (MC + S)

and control rats (MC + H2O) (p b 0.1). However, the combined
treatment with S and the extracts led to more profound results,
since the increase of locomotor activities was observed and the
differences were statistically significant (RE + S vs. MC + H2O,
p b 0.01; RA + S vs. MC + H2O, p b 0.05; huperzine A + S vs.
MC + H2O, p b 0.01). It should be stressed that no significant
differences were found between the combined (RE + S,
RA + S, huperzine A + S) and the S-treated animals (p N 0.1),
therefore the effect was produced mainly by S injection, which
was confirmed when compared with the substance + H2O
proper groups (RE + S vs. RE + H2O, p b 0.01; RA + S vs.
RA + H2O, p b 0.05; huperzine A + S vs. huperzine A + H2O,
p b 0.1).
A one-way ANOVA analysis revealed significant differences when the rats' locomotor activity was expressed as the
number of their climbs (vertical activity) (F(7, 91) = 2.57;
p b 0.05; Table 1). The stimulation effect was produced by a
single S administration, but the detailed post-hoc analysis
showed no statistically significant differences between the
S-treated animals (MC + S) and control rats (MC + H2O)
(p b 0.1). However, the combined S and RA extract led to
more profound results: an increase of vertical activities was
observed and some differences were statistically significant
(RE + S vs. MC + H2O, p b 0.1; RA + S vs. MC + H2O,
p b 0.01; RE + S vs. RE + H2O, p b 0.05; RA + S vs.
RA + H2O, p b 0.01). As for the horizontal activities the
effects were produced mainly by the combined S injection. In
the case of RA, the combined treatment with RA and S

4.1.2. Motor coordination

A one-way ANOVA analysis revealed significant differences in motor coordination of rats expressed as their exit
time from the cylinder (F(7, 91) = 6.63; p b 0.001; Table 1).
Detailed analysis showed that scopolamine produced the
prolongation of exit time, but the difference was not
statistically significant (MC + S vs. MC + H2O, p b 0.1).
Moreover, the multiple administration of RA, huperzine A
and RE treatment did not affect significantly this paradigm of
rats. However, the combined S and extract or RA led to more
profound results, since the prolongation of exit time was
observed when compared with control rats (RE + S vs.
MC + H2O, p b 0.01; RA + S vs. MC + H2O, p b 0.01), or
with the extract or RA-treated animals without S injection
(RE + S vs. RE + H2O, p b 0.05; RA + S vs. RA + H2O,
p b 0.01). The observed effects were not only produced by
the presence of S, since the time of exit was significantly
prolonged in comparison with S treated rats (RE + S vs.
MC + S, p b 0.01; RA + S vs. MC + S, p b 0.01).
4.1.3. Long term memory
A one-way ANOVA analysis revealed significant differences in long term memory after using a passive avoidance
test after 24 h from footshock (F(7, 89) = 4.88; p b 0.001;

Table 1
Effects of multiple treatment (28x) of Rosmarinus officinalis extract on behavioral and cognitive activities in rats.
Groups

Locomotor activity
Spontaneous activity

MC + H2O
Huperzine A + H2O
RA + H2O
RE + H2O
MC + S
Huperzine A + S
RA + S
RE + S

Number of climbs

Motor coordination

Long-term memory

Short-term memory

Exit timea

Passive avoidance test [latency]

Object recognition test

[number of impulses/5 min]

[number/5 min]

[s]

387
(20n)
484
(10)
410
(10)
356
(9)
530
(20)2
645
(10)
612
(10)
670
(10)

60 8
(20)
95 18
(10)
57 20
(10)
48 12
(9)
75 14
(20)
83 16
(10)
145 43***,+++,
(10)
122 23*,++,
(10)

18
(20)
18
(10)
24
(10)
28
(9)
32
(20)
29
(10)
52
(10)
53
(10)

27
42*
65
52
54*
83***
72**

47***,

3
5
6
6
5*
7
5***,

++

4***,++

after 24 h [s]

ratio ORb

51 14
(20)
146 19***
(10)
65 25
(10)
85 29
(9)
12 3**
(20)
58 20++
(10)
35 15
(10)
58 24++
(10)

0.39
(20)
0.37
(10)
0.46
(9)
0.27
(9)
0,30
(20)
0.23
(10)
0.41
(10)
0.49
(10)

0.07
0.08
0.09
0.10
0.07
0.06
0.09
0.06+

n - number of rats
values expressed as mean SEM
RE - extract from Rosmarinus officinalis leaves (200 mg/kg, p.o.)
RA - rosmarinic acid in a dose of 10 mg/kg, p.o.
Huperzine A - in a dose of 0.5 mg/kg, p.o.
S - scopolamine in a dose of 0.5 mg/kg, i.p.
A - exit time in the chimney test
B - ratio OR = (B A*)/(B + A*), where: B - the time spent exploring the novel object (session II); A* - the time spent exploring the familiar object during
session II (for details, see Materials and Methods)
***,**,* - statistical difference vs. control (MC + H2O), p b 0.01, p b 0.05 or p b 0.1, respectively
+++, ++, +
- statistical difference vs. scopolamine-treated group (MC + S), p b 0.01, 0.05 or p b 0.1, respectively
, , statistical difference vs. proper substance + vehicle treated-group (Huperzine A + H2O, RA + H2O or RE + H2O), p b 0.01, 0.05 or p b 0.1,
respectively

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M. Ozarowski et al. / Fitoterapia 91 (2013) 261271

significance was shown only for RE as compared with the

control group (vs MC + H2O, p b 0.05) (Table 2). Similarly, RE
and RA significantly elevated the BuChE activity in the
hippocampus by 61% (vs MC + H2O, p b 0.01) and by 81% (vs
MC + H2O, p b 0.01), respectively. On the contrary, huperzine
A did not change the BuChE activity in the hippocampus.

Table 1). It was shown that the strongest effect leading to an

improvement of this paradigm was produced by huperzine A
when compared with control animals (p b 0.01), whereas the
RA and RE marginally improved long-term memory, but the
results were statistically non-significant in comparison with
the control group (MC + H2O). However, the administration
of scopolamine to rats significantly decreased the latency
time of passive avoidance task (MC + S vs. MC + H2O,
p b 0.05). After RE or huperzine A combined treatment with
S an improvement of long term memory was observed
(RE + S vs. MC + S, p b 0.05; huperzine A vs. MC + S,
p b 0.05). Therefore, it can be concluded that administration
of RE or huperzine A overcomes the effects shown by S.

4.3. mRNA AChE and BuChE gene expression level in rat brain
A one-way ANOVA analysis revealed significant differences
in gene expression of mRNA AChE both in the cortex and the
hippocampus (cortex: ANOVA F(3,28) = 3.21, p b 0.05; hippocampus: ANOVA F(3,34) = 7.80, p b 0.001). As shown in
Table 3 the multiple treatment of RE produced a statistically
significant increase of the relative mRNA AChE gene expression
level by 65% in the hippocampus (vs. MC + H2O, p b 0.01)
without any effect on the cortex. Moreover, it was demonstrated that administration of huperzine A significantly
decreased the relative mRNA AChE expression level by 44% in
the cortex when compared with the control (p b 0.05). RA
administration did not affect mRNA AChE expression neither in
the cortex nor in the hippocampus.
There were also significant differences between the
relative values of mRNA BuChE expression (cortex: ANOVA
F(3,26) = 8.70, p b 0.001; hippocampus: ANOVA F(3,31) =
6.42, p b 0.001). Further analysis showed that RE treatment
led to a decrease in the mRNA BuChE expression level by 59%
in the cortex (vs. MC + H2O, p b 0.05) and, simultaneously, a
statistically significant increase of the transcript level by 124%
in the hippocampus (vs. MC + H2O, p b 0.01) (Table 3). On the
contrary, RA treatment produced an elevation of the transcript
level in the cortex (88%, vs. MC + H2O, p b 0.01) and a
decrease in the mRNA BuChE expression in the hippocampus
(37%, p b 0.05). The prolonged huperzine A administration
resulted in a decrease of the transcript level in the cortex (49%,
vs. MC + H2O, p b 0.05), with no changes in the hippocampus.

4.1.4. Short term memory

The results of the object recognition test showed that the
administration of the compounds or extract did not affect the
rats' short term memory (one way ANOVA F(7, 90) = 1.12,
p N 0.1). However, the detailed post-hoc analysis showed an
improvement of rats' short term memory after RE + S
treatment when compared with S-treated animals, but the
difference was non-significant (RE + S vs. MC + S, p b 0.1).
4.2. Acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE) activities in rat brain
A one-way ANOVA revealed significant differences in the
activity of AChE both in the cortex and the hippocampus
(cortex: ANOVA F(3,34) = 8.82, p b 0.001; hippocampus:
ANOVA F(3, 34) = 10.8, p b 0.001). It was found out that RE
showed an inhibition of AChE activity in the frontal cortex by
55% (p b 0.01) and in the hippocampus by 72% (p b 0.01) after
28 days of treatment when compared with control rats
(MC + H2O) (Table 2). The effect of RE was similar to the that
of RA and huperzine A, since RA had an ability to inhibit AChE in
the frontal cortex (38%, p b 0.01) and in the hippocampus (46%,
p b 0.01) vs. MC + H2O, whereas huperzine A showed less
inhibition of AChE activity in comparison to RE by 48%
(p b 0.01) and 47% (p b 0.01) in the cortex and the hippocampus, respectively.
There were also significant differences between the values
of BuChE activities (cortex: ANOVA F(3,35) = 2.84, p b 0.05;
hippocampus: ANOVA F(3,35) = 8.59, p b 0.001). Contrary to
the effects on AChE, RE and RA increased the BuChE activities in
the frontal cortex by 32% and 16%, respectively, but statistical

4.4. Phytochemical prole of ethanol extract of R. ofcinalis

leaves
4.4.1. Flavonoids and polyphenolic acids
It was found that the major compounds in hydro-ethanolic
R. officinalis leaf extract established by HPLC were RA (5.46%),
carnosic acid (2.48%) and carnosol (1.32%) of the 45 identified
chemical compounds (Table 4, Fig. 1). Moreover, the total

Table 2
Effects of extract from leaves of Rosmarinus officinalis (200 mg/kg, p.o.) in chronic treatment on AChE and BuChE activities in rat brain.
Groups

AChE [nmol ATCh/min/mg protein]

MC + H2O
Huperzine A + H2O
RA + H2O
RE + H2O

361
188
224
163

Cortex

49
18***
21***
17***

BuChE [nmol BuTCh/min/mg protein]

Hippocampus

Cortex

Hippocampus

449
238
242
126

69
58
80
92

54
52
97
86

74
22***
26***
18***

Value expressed as mean SEM (n = 810)

AChE - acetylcholinesterase
BuChE - butyrylcholinesterase
Huperzine A - in a dose of 0.5 mg/kg, p.o.
RA - rosmarinic acid (10 mg/kg, p.o.)
RE - extract from the leaves of Rosmarinus officinalis
***,** - statistical difference vs. control (MC + H2O), p b 0.01 or p b 0.05, respectively

12
7
8
4**

8
4
9***
9***

M. Ozarowski et al. / Fitoterapia 91 (2013) 261271

267

Table 3
Effect of extract from the leaves of Rosmarinus officinalis (200 mg/kg, p.o.) chronic treatment on mRNA AChE and BuChE gene expression in rat brain.
Groups

MC + H2O
huperzine A + H2O
RA + H2O
RE + H2O

mRNA AChE

mRNA BuChE

Cortex [%]

Hippocampus [%]

Cortex [%]

Hippocampus [%]

100 12
56 6**
117 21
99 14

100 11
85 5
109 10
165 20**

100 18
42 12**
188 36***
41 8**

100 11
102 11
63 11**
224 55**

Values expressed as mean SEM (n = 610)

Control group (MC + H2O) defined as 100%
AChE - acetylcholinesterase
BuChE - butyrylcholinesterase
Huperzine A - in a dose of 0.5 mg/kg, p.o.
RA - rosmarinic acid (10 mg/kg, p.o.)
RE - extract from the leaf of Rosmarinus officinalis
***,** - statistical difference vs. control (MC + H2O), p b 0.01 or p b 0.05, respectively

polyphenols and flavonoid content of RE was determined with

the use of FolinCiocalteu assay. The extract showed 23.58% of
gallic acid. The total hydroxycinamic derivatives expressed as
rosmarinic acid by HPLC was 12.95%.
4.4.2. Essential oil composition
The GC/FID analysis showed that the extract comprised 18
components (Fig. 2). The main ones included 1.8-cineole
(32.7%), alfa-pinen (9.72%), camphor (7.91%), camphene
(2.41%), beta-pinene (2.14%), myrcene (2.13%), limonene
(1.75%), trans-caryophyllene (1.59%), alfa-terpineol (1.53%),
borneol (1.39%), eugenol (1.10%), gamma-terpinene (0.69%),
linalool (0.77%), p-cymene (0.48%), bornyl acetate (0.27%),
terminen-4-ol (0.23%), thymol (0.06%) and sabinen (0.04%).
5. Discussion
In folk medicine, R. officinalis is used for the prevention and
treatment of cognitive impairment [2022] but so far little
evidence is yet available with regards mechanisms of extract
action that are potentially relevant to cognitive function.
The present study investigated the influence of subchronic
(28-fold) administration of standardized 50% EtOH extract of
R. officinalis (200 mg/kg, p.o.) on scopolamine impaired shortterm and long-term memory. The results were compared with
the activity of cholinesterases (AChE and BuChE) as well as
with AChE and BuChE gene expression level in the cortex and
hippocampus of the rat brain.
In our study huperzine A was used in animal model as a
positive control to comparison with results of extract from
leaves of R. officinalis. This novel alkaloid isolated from the
Chinese herb Huperzia serrata, is not only a potent and reversible
acetylcholinesterase (AChE) inhibitor but also possessed the
antioxidant and neuroprotective activities within the cerebral
cortex and hippocampus [23]. Moreover, huperzine A inhibited
BuChE [24]. Huperzine A has attracted considerable attention
worldwide because of its unique chemical structure, its memory
enhancing effects observed in both animal and clinic trials, and
its low toxicity [23,25]. Currently, this natural compound is a
promising drug candidate for Alzheimer's disease [26]. On the
other hand, scopolamine (S), a muscarinic antagonist that
induces central cholinergic blockade is a chemical substance for
induction of memory impairment in animal model because it
produces a reversible and well-described impairment in both

maintaining attention and processing of information, and the

acquisition of new knowledge in rodents and in human [27].
Thereby, S-induced amnesia has been proposed as a model for
dementia [28], has been used as an experimental model for
Alzheimer's disease [29] and was used in many other studies
coupled with the assessement of cognitive functions in rodents
[3,6,30]. Actually, S in our experiment showed an opposite effect
in comparison with extract from R. officinalis leaves. It was
observed that the administration of S to rats significantly
decreased the latency time of passive avoidance task suggesting
that S impaired a long term memory in animals. Additionally, it
was observed that S diminished activity of AChE in the frontal
cortex and hippocampus by 50% and 45%, and S decreased also
activity of BuChE in the frontal cortex by 62% and by 47% in
hippocampus (data not shown).
The multiple administration of RE significantly increased
the step-through latency especially in scopolamine-treated
rats, what is in line with the results of Zanella et al. [31]
showed that hydroalcoholic extract of R. officinalis (150 and
300 mg/kg) improved learning and memory processes of
mice in inhibitory avoidance task.
The effect seemed to be specific since RE did not produce
sedative activity when RE was administered alone or in
combination with S. Furthermore, S showed a tendency to
increase the rats' spontaneous activity and number of climbs,
and the effects were significant in RE + S rats. It is probably
due to the fact that S in the low dose used in this study does
not act as a CNS depressant and stimulates exploratory
behavior in rodents by muscarinic antagonism, facilitating
the release of an excitatory neurotransmitter, i.e. acetylcholine (turn-over effect) [32]. S treatment led to reduced motor
coordination, and especially the combination with the extract
showed a strong significant attenuation of this paradigm;
however, it is also claimed that S demonstrates a lack of
correlation between motor skills and learning abilities [33].
Therefore, the answer to the question whether the impairment of motor coordination and simultaneous increasing of
locomotor activity of rats produced by S can affect the
memory results remains open.
Due to the fact that the direction and strength of the
behavioral and cognitive changes after application of RE
were similar to huperzine A both in nonscopolamine- and
scopolamine-treated rats, it can be presumed that the mechanism of action of the extract could be comparable to a reference

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M. Ozarowski et al. / Fitoterapia 91 (2013) 261271

Table 4
Metabolites detected in rosemary extracts by HPLC-UV-MS.
Peak number

Rt (min)

Molecular weight

Major fragments (m/z)

Ionization mode

Compound identified

Reference

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45

6,02
8,1
10
16,7
16,9
17,1
17,2
17,2
17,4
17,3
17,5
17,6
18,4
18,8
19,2
19,4
21,6
22
22,7
23,1
23,2
23,3
23,4
23,5
23,6
23,9
24,6
24,9
25
25,4
27,5
27,5
29,1
30
33,4
36,2
42,5
42,7
44,1
45,7
45,8
46,5
47
48
51

306
388
372
594
684
478
462
624
598
640
610
462
534
522
478
578
360
462
550
684
640
476
492
610
504
684
504
624
564
504
546
672
546
314
346
346
344
360
330
344
344
374
316
332
346

305,
387,
371,
593,
683,
477,
461,
625,
597,
641,
611,
463,
533,
521,
477,
577,
359,
461,
549,
683,
641,
477,
493,
611,
503,
683,
503,
623,
563,
503,
545,
671,
545,
313,
345,
345,
343,
359,
329,
343,
343,
373,
315,
331,
345,

N
N
N
N
N
N
N
P
N
P
P
P
N
N
N
N
N
N
N
N
P
P
P
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N

Gallocatechin
Medioresinol
Caffeic acid derivative
Luteolin 7-O-rutinoside
Caffeic acid derivative
Isorhamnetin-3-O-glucoside
Luteoline 7-O-glucuronide
Isorhamnetin-3-O-rutinoside
Eriocitrin
Isorhamnetin-3-O-di-glucoside
Luteoline 7-O-di-glucoside
Homoplantaginin
Caffeic acid derivative
Rosmanoylglucoside
Cirsimaritin 4'-O-glucoside
Apigenin rutinoside
Rosmarinic acid
Scutellarein 7-glucuronide
Medioresinol caffeate
Caffeic acid derivative
6- hydroxyluteoline glucosylglucuronide
Hispidulin 7-O-glucuronide
Tricin glucoside
6- hydroxyluteoline rutinoside
Luteolin 3'-O-(O-acetyl)--D-glucuronide I
Rosmarinic acid derivative
Luteolin 3'-O-(O-acetyl)--D-glucuronide II
Feruloylnepitrin
Caffeoylmedioresinol
Luteolin 3'-O-(O-acetyl)--D-glucuronide III
Luteolin di-O-acetyl--D-glucuronide II
Flavone derivative
Luteolin di-O-acetyl--D-glucuronide II
Cirsimaritin
Rosmanol
Epirosmanol
Rosmadial
Rosmanol methyl ether
Carnosol
Galdosol
Safficinolide
7-ethoxyrasmanol
Rosmaridiphenol
Carnosic acid
Methylcarnosate

13
13
NI
12
NI
13
12
NI
9
NI
NI
12
NI
NI
12
14
12
12
NI
NI
NI
NI
NI
9
12
NI
12
12
NI
12
NI
NI
NI
13
7
7
7
7
7
12
12
12
12
7
7

225,
207,
249
285,
665,
315,
285
479,
311
479,
287
301,
323,
359,
315,
269
161
285,
387,
653,
465,
301,
331,
303
399,
521,
285,
315,
387,
443,
441,
509,
485,
298,
301,
301,
299,
283,
285,
315,
299,
283,
285
287,
301,

207
163, 145, 109
243, 175
397, 353
299.6
317, 302
317, 302
286
179
161
153

255
207, 163
621, 353, 161
303
286
316
283, 255
359, 197, 161
243
299.6
207
285, 255
384, 285, 255
311, 283
381, 285, 255
283, 255
283
283
227
211
270, 203
287
281
227
244
286

In ionization mode column: N indicates fragmentation of molecules obtained in negative ionization mode and P indicates fragmentation of molecules obtained in
positive ionization mode. Number of peaks correlates with numbers of peaks on chromatogram UV at Fig. 1.
NI - compound not found in literature

compound used as a potent acetylcholinesterase inhibitor

[34]. Although cholinesterase inhibitors can attenuate the
scopolamine-induced hyperactivity, in order to obtain this effect
relatively high doses of the inhibitors (i.e. physostigmine) are
required [35]. Therefore, it can be said that both the doses of
huperzine A and RE used in the present study are too low to
produce such an effect.
RA (5.46% in this study) is one of the most important
phenolic acids whose content is relatively high in RE. It was
found that RA in the dose of 10 mg/kg b.w. (p.o.) did not
affect either short or long term memory. The results are in
agreement with those reported by Pereira et al. [36] who
demonstrated that administration of the RA to rats 30 min
before the training session for the step-down inhibitory
avoidance test did not induce any effect, indicating that this

compound (in the doses of 1, 2, 4 or 8 mg/kg, i.p.) did not

affect short- and long-term memory. Moreover, we observed
that the repeated administration of RA in non-scopolamine
treated rats did not produce any changes of locomotor
activity, similarly to RE or huperzine A. This is in line also
with the observations of Pereira et al. [36].
The present study demonstrated that RE, huperzine A and
RA inhibited the AChE activity in the rats' frontal cortex and
hippocampus. On the other hand, RE showed a stimulatory
effect on BuChE in both parts of rat brain, whereas RA
produced such an action in the hippocampus only. In this
condition huperzine A did not affect the BuChA activity of
rats. The effect produced by huperzine A is in agreement with
the results by other authors, that the compound is a potent
reversible inhibitor of AChE over BuChE, which can be

M. Ozarowski et al. / Fitoterapia 91 (2013) 261271

269

Fig. 1. Chromatogram UV of rosemary extract obtained at 254 nm with peaks indentified by HPLC-UV-MS.

inhibited 1000-fold less than AChE [37]. The effects of RE and

RA on AChE are less clear. It has been known from in vitro
studies that an aqueous and methanol extract of R. officinalis
leaves inhibited the AChE activity by 12% and 17% at 0.1 mg/
mL concentration, respectively [38]; however Orhan et al. [6]
showed that different extracts of R. officinalis (methanol,
petroleum ether, chloroform, ethyl acetate) were not able to
inhibit AChE and BuChE at 0.2 and 0.5 mg/mL concentration.

Therefore, it can be speculated that RE inhibitory activity

against AChE or/and BuChE is probably linked both with the
way of its preparation and conditions of the experimental
study.
The next step in our study was to determine a possible
correlation between the AChE and BuChE mRNAs and their
protein quantitative changes in the brain homogenates
(frontal cortex and hippocampus) in rats due to the fact

Fig. 2. GC-FID profile of essential oil from the leaves of R. officinalis. The following compounds present in extracts of Rosmarinus officinalis leaves: alpha-pinene
(retention time: 3.56 min), camphene (4.43), beta-pinene (5.28), myrcene (6.98), alpha-terpineol (7.32), limonene (8.32), 1,8-cyneol (8.77), gamma-terpinene
(10.71), p-cymene (12.16), sabinen (24.48), camphor (26.54), linalol (29.41), bornyl acetate (30.19), trans-caryophyllene (30.77), terminen-4-ol (32.38), borneol
(37.85), eugenol (53.92), thymol (60.45).

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M. Ozarowski et al. / Fitoterapia 91 (2013) 261271

that the molecular mechanism of RE activity or its bio-active

compounds in any model organism or in humans is still
unknown.
We observed a surprisingly different AChE and BChE mRNA
expression profile in the frontal cortex and the hippocampus
tissues in the animals receiving RE and RA, while the activity of
the two enzymes in the cortex and hippocampus, under the
influence of the extract, did not differ significantly.
In our opinion, the differences in the relative mRNA
expression levels of AChE and BuChE in the cortex and the
hippocampus of experimental animals in comparison to their
activity can be explained by their diverse location, structure
and function as well as by their quite complicated and not
fully understood, molecular mechanisms regulating their
activities.
The discrepancy between the activity and mRNA expression
may be due to the location of the AChE enzyme and the
prevalence of its different molecular forms [39]. It is suggested
that various posttranscriptional mechanisms may be responsible for AChE's quantitative, spatial and temporal variations
[39,40].
The differences between the protein activity and expression
level of mRNA may be also caused by molecular mechanisms
occurring as changes in the nucleic acid level. Alternative
splicing leading to the formation of numerous enzyme variants
is an example of molecular regulation presenting an attempt to
clarify the discrepancy between the low levels of protein and
observed different mRNA expression [41]. The present study
merely assessed the overall total pool of mRNA and the overall
level of activity of AChE enzyme. Consequently, further
research will be necessary to assess the activity and separate
expression of mRNA of different isoforms of the enzyme for a
thorough evaluation of AChE adjustment.
Among the molecular mechanisms underlying the changes
in expression levels there are also transcription factors.
Researchers suggested the existence of a feedback mechanism
through which the AChE gene is activated by cholinergic
neurotransmission, possibly leading to an increased formation
of AChE protein and accelerated degradation of acetylcholine at
cholinergic synapses [42].
As mentioned above, in the light of current knowledge, there
is no clear evidence explaining different responses of these two
enzymes under the influence of RA and RE. This may be partially
due to the fact that the exact function of the second studied
enzyme BuChE is not fully understood. It is possible that the
observed differentiation of BuChE mRNA expression levels in
the frontal cortex and the hippocampus for RE and RA may be
due to the differences of butyrylcholinesterase localization and
substrate affinity [43]. Since in our experiment we have carried
out a quantitative analysis of AChE and BuChE transcripts in
brain homogenates of tested animals, rather than in individual,
isolated cell fractions, therefore the obtained results constitute
an overall picture of both studied genes transcriptional
changes occurring in the brain areas of studied animals under
the influence of the tested substances and extract.
In summary, at present it is not clear how to explain both
mechanisms of action of RE linked with enhancement of long
term memory and its possible inhibitory action of AChE.
Therefore, it can be stipulated that not only the presence of
RA is involved in its action, but also many other compounds.
In previous studies [6] were found out that components of

essential oils of R. officinalis could inhibit the AChE and BuChE

activity in vitro. It was concluded that anticholinesterase
activity of rosemary essential oil most likely depends on a
synergic mechanism between a number of oil components. On
the other hand, polyphenols still constitute a promising source
of new drugs and there is a high interest in understanding their
mechanisms [44,45].
It's well known that extract of leaves of R. officinalis includes
several biologically active compounds, most notably phenolic
acids, phenolic diterpenes, pentacyclic triterpenes, flavonoids
and essential oil [79,1214], therefore it can be assumed that
its observed pharmacological action is due to the combined
action of the several constituents. In our study it was calculated
that rosmarinic acid, carnosic acid and carnosol of the 45
identified chemical compounds were in the highest concentrations in studied extract of R. officinalis, therefore their
possible interactions at the pharmacological activity can be
taken into consideration.
6. Conclusion
The subchronic administration of complex RE led to an
improvement of long-term memory of rats, which can be
partially explained by its inhibitory action on AChE activity in
rats' frontal cortex and hippocampus. It seems that the RE
activity represents a possible option for preventive treatment
against the risk of some neurodegenerative diseases.
Acknowledgments
This study was carried out within the framework of a
research project no. N 405417836 financed by the Polish
Ministry of Science and Higher Education. Authors declare no
conflict of interest with any financial organization regarding
the material discussed in the manuscript.
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