Chemico-Biological Interactions 237 (2015) 47–57

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Rosemary tea consumption results to anxiolytic- and anti-depressant-

like behavior of adult male mice and inhibits all cerebral area and liver
cholinesterase activity; phytochemical investigation and in silico studies
Anastasia-Varvara Ferlemi a,1, Antigoni Katsikoudi a,2, Vassiliki G. Kontogianni b, Tahsin F. Kellici b,f,
Grigoris Iatrou d, Fotini N. Lamari e, Andreas G. Tzakos b,c,⇑, Marigoula Margarity a,⇑
a
Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, 26504 Rio, Greece
b
Department of Chemistry, Section of Organic Chemistry and Biochemistry, University of Ioannina, GR-45110 Ioannina, Greece
c
CancerBiobank Center, University of Ioannina, GR45110 Ioannina, Greece
d
Plant Division, Department of Biology, University of Patras, 26504 Rio, Greece
e
Laboratory of Pharmacognosy & Chemistry of Natural Products, Department of Pharmacy, University of Patras, 26504 Patras, Greece
f
Department of Chemistry, Laboratory of Organic Chemistry, National and Kapodistrian University of Athens, Panepistimioupolis, Zografou 15771, Greece

a r t i c l e i n f o a b s t r a c t

Article history: Our aim was to investigate the possible effects of regular drinking of Rosmarinus officinalis L. leaf infusion
Received 22 January 2015 on behavior and on AChE activity of mice. Rosemary tea (2% w/w) phytochemical profile was investigated
Received in revised form 24 March 2015 through LC/DAD/ESI-MSn. Adult male mice were randomly divided into two groups: ‘‘Rosemary-treated’’
Accepted 12 April 2015
that received orally the rosemary tea for 4 weeks and ‘‘control’’ that received drinking water. The effects
Available online 21 April 2015
of regular drinking of rosemary tea on behavioral parameters were assessed by passive avoidance, ele-
vated plus maze and forced swimming tests. Moreover, its effects on cerebral and liver cholinesterase
Keywords:
(ChE) isoforms activity were examined colorimetricaly. Phytochemical analysis revealed the presence
Rosmarinus officinalis
Cholinesterase
of diterpenes, flavonoids and hydroxycinnamic derivatives in rosemary tea; the major compounds were
Step-through passive avoidance test quantitatively determined. Its consumption rigorously affected anxiety/fear and depression-like behavior
Elevated plus maze test of mice, though memory/learning was unaffected. ChE isoforms activity was significantly decreased in
Forced swimming test brain and liver of ‘‘rosemary treated’’ mice. In order to explain the tissue ChE inhibition, principal
Molecular docking component analysis, pharmacophore alignment and molecular docking were used to explore a possible
relationship between main identified compounds of rosemary tea, i.e. rosmarinic acid,
luteolin-7-O-glucuronide, caffeic acid and known AChE inhibitors. Results revealed potential common
pharmacophores of the phenolic components with the inhibitors. Our findings suggest that rosemary
tea administration exerts anxiolytic and antidepressant effects on mice and inhibits ChE activity; its main
phytochemicals may function in a similar way as inhibitors.
Ó 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
The consumption of herbals and herbal supplements for both
the alleviation of the diseases’ symptoms and the prevention is
⇑ Corresponding authors at: Cancer Biobank Center & Dept. of Chemistry,
Section of Organic Chemistry and Biochemistry, Univ. of Ioannina, GR-45110
Ioannina, Greece. Tel.: +30 2651008387 (A.G. Tzakos). Laboratory of Human and
Animal Physiology, Dept. of Biology, University of Patras, Greece. Tel.: +30 2610
997430; fax: +30 2610 969272 (M. Margarity).
E-mail addresses: agtzakos@gmail.com (A.G. Tzakos), margar@upatras.gr
(M. Margarity).
1
Present address: Laboratory of Pharmacognosy & Chemistry of Natural Products,
Department of Pharmacy, University of Patras, 26504 Patras, Greece.
2
Present address: Barts and the London School of Dentistry & Medicine, Blizard
Institute, Center of Neuroscience and Trauma, London, UK.
renowned in all parts of the world since the ancient times.
Nowadays, based on traditional remedies, several studies demon-
strate that nutritional and herbal supplementation is an effective
method for treating anxiety and anxiety-related conditions [1],
although there is a lack of rigorous clinical studies in this area [2].
Rosmarinus officinalis L. (Labiatae, rosemary), mostly naturalized
from cultivation, but locally native in Greece, is a common culinary
herb cultivated in many parts of the world. It has been attributed a
number of therapeutic applications in folk medicine systems such
as against dyspepsia and mild spasmodic disorders of the gastroin-
testinal tract, minor muscular and articular pain and minor periph-
eral circulatory disorders [3], as well as respiratory and
inflammatory diseases [4]. Moreover, rosemary is listed among
European herbs that have been pan-culturally used in order to

http://dx.doi.org/10.1016/j.cbi.2015.04.013
0009-2797/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
48 A.-V. Ferlemi et al. / Chemico-Biological Interactions 237 (2015) 47–57
alleviate cognitive deficits [5], whereas, an ethnopharmacological
use of R. officinalis in the treatment of depression, among other
uses, was also reported by Heinrich et al. [6].
The implication of the cholinergic system in learning and mem-
ory process is well established [7–9]. Even though its implication
in anxiety-like and depression-like behavior is also acknowledged
[10–12], several reports show controversial results, indicating
the complexity of the behavioral mechanisms. In the study by
Mineur et al. [13], experiments in mice using AChE inhibitors, such
as physostigmine, demonstrated an increase in anxiety- and
depression-like behaviors that were reversed using muscarinic
and nicotinic antagonists. Several AChE inhibitors, such as donepe-
zil, rivastigmine, galantamine and memantine have been approved
by EMA for the treatment of dementia and Alzheimer’s disease
(AD), diseases associated to the degeneration of the cholinergic
system [14,15]. Rozzini et al. [16] demonstrated that acetyl-
cholinesterase inhibitor therapy in depressed AD patients, alleviated
the depressive symptoms and this improvement was independent
of cognitive enhancement, indicating the multi-targeted positive
result of AChE inhibition. However, conventional treatment modal-
ities are hindered by adverse effects and produce only a partial
remission [17]; thus, there has been renewed interest in medicinal
plants, which may have comparable efficacy to prescription medica-
tions, while lacking their severe side effects [18].
In this frame, we assessed the protective potential of regular
drinking of rosemary infusion. Specifically, the aim of the present
study was to investigate the potential protective effects of the con-
sumption of rosemary leaf infusion on several behavioral aspects
on adult male mice, i.e. anxiety/fear, depression-like behavior
and memory/learning, and on cerebral and hepatic ChE isoforms
activity. Finally, using principal component analysis, pharma-
cophore alignment and molecular docking, we explored the
relationship between caffeic acid, carnosic acid, rosmarinic acid,
luteolin-7-O-glucuronide with known AChE inhibitors.
2. Materials and methods
2.1. Preparation of the 2% w/v infusion of R. officinalis and extraction
R. officinalis (rosemary) leaves were collected from plants
cultivated and grown in the garden of the Department of Biology,
in the University Campus of Patras, during September 2012 and
identified by Prof. Gregorios Iatrou. A voucher specimen (No.
UPA150912) was deposited at the Herbarium (UPA) of the
Department of Biology. For infusion preparation, 2 g of rosemary
leaves were placed into 100 mL of hot water, which was further
boiled at 100 °C for 1 min. The leaves were then left to steep for
5 min, filtered and made to the volume of 100 mL. The infusion
was strained, left at room temperature to cool down and given to
the animals.
2.2. Determination of total phenolics, flavonoids and monomeric
anthocyanins of rosemary infusion
Total phenolics were measured with the Folin–Ciocalteau
reagent method [19]. The total polyphenolic content was
expressed as gallic acid equivalents (GAE), using a standard curve
with 0.1–1.0 mg/mL gallic acid. The data are expressed as GAE
per 100 mL of infusion.
The total flavonoid content was determined according to the
aluminum chloride colorimetric method [20] as follows: in a 96
well – plate, 16 lL of the rosemary infusion were mixed with
40 lL 95% alcohol, 5 lL 10% aluminum chloride (AlCl3), 5 lL 1 M
potassium acetate (CH3COOK) and 75 lL distilled water. After
incubation for 40 min at room temperature, the reaction mixture
absorbance was measured at 405 nm against distilled water blank.
Standards (5–200 lg/mL) were prepared using quercetin. Total
flavonoids were expressed as quercetin equivalents (QE) per
100 mL of rosemary infusion.
w?>Total monomeric anthocyanins were estimated using the
pH-differential method of [21]. According to the method, the absor-
banceoftwodilutionsoftheinfusion,onewith0.025 Mpotassiumchlo-
ridebuffer,pH1.0andtheotherwith0.4 Msodiumacetatebuffer,pH4.5,
were measured at 510 and 700 nm, respectively. Total monomeric
anthocyanin content was expressed as cyanidin-3-glucoside equiva-
lentsper100 mLofrosemarybeverage.
2.3. LC–MS analysis and quantitative determination of the major
phenolic compounds
The LC–MS analysis was performed according to [22]. All
LC–MSn experiments were performed on a quadrupole ion trap
mass analyzer (Agilent Technologies, model MSD trap SL) retro-
fitted to a 1100 binary HPLC system equipped with a degasser,
an autosampler, a diode array detector and an electrospray ioniza-
tion source (Agilent Technologies, Karlsruhe, Germany). All hard-
ware components were controlled by Agilent Chemstation
Software.
Rosemary infusion was freeze dried and an aqueous extract was
obtained. Aqueous rosemary extract was dissolved in 50–50% v/v
acetonitrile–water (1 mg dry extract/mL). A 10 lL aliquot was fil-
tered (0.45 lm) and injected into the LC–MS instrument.
Separation was achieved on a 25 cm  4.6 mm i.d., 5 lm Zorbax
Eclipse xdb-C8 analytical column, at a flow rate of 0.6 mL min 1,
using as solvent A (water/formic acid, 99.9:0.1 v/v) and solvent B
(acetonitrile). The gradient used for the analysis of extracts was:
0–20 min, 80–40% A; 20–25 min, 40–25% A; 25–40 min, 25–15%
A; 40–45 min, 15–80% A. Chromatograms were acquired at 254,
284, and 330 nm.
Both precursor and product ion scanning of the phenolic com-
pounds was monitored between m/z 50 and m/z 1000 in negative
polarity. The ionization source conditions were as follows: capil-
lary voltage, 3.5 kV; drying gas temperature, 350 °C; nitrogen flow
and pressure, 10 L min 1 and 50 psi, respectively. Maximum accu-
mulation time of ion trap and the number of MS repetitions to
obtain the MS average spectra were set at 30 ms and 3, respec-
tively. Authentic standard compounds used for comparison rea-
sons were quinic acid (98%, Aldrich, Steinheim, Germany),
carnosol and carnosic acid (98% and 91%, respectively, Sigma,
Steinheim, Germany).
Quantitative analysis was performed by HPLC method using a
diode array detector with the authentic standards that are com-
mercially available; rosmarinic acid (95%, Fluka, Steinheim,
Germany), caffeic acid (98%, Aldrich, Steinheim, Germany) that
was used to quantitate caffeic acid derivative and
luteolin-7-O-glucoside (98%, Extrasynthese, France) that was used
to quantify luteolin-7-O-glucuronide. All compounds were quanti-
tated at 330 nm.
2.4. Animals
Male 3–4 month-old Balb-c mice (30–40 g bw), were kept in
polyacrylic cages (n = 16/group) and housed in a room under con-
trolled temperature of 24–26 °C, relative humidity between 50–
60% and with 12 h alteration of light and dark cycles. The adult
mice were randomly divided into two groups: (i) a control group
which consumed drinking water for 4 weeks (200 mL) and (ii) a
Rosemary-treated group which consumed the R. officinalis infusion
(2% w/v/day for 4 weeks). Practically, rodents received daily
130 mg dry extract/kg of body weight. This particular dosage of
rosemary tea is the one usually recommended for preparation of
 A.-V. Ferlemi et al. / Chemico-Biological Interactions 237 (2015) 47–57
herbal teas in phytotherapy [23]. The consumption of food and of
water/infusion was recorded daily and mouse body weight was
measured every 7 days. After behavioral tests, mice were killed
with decapitation after light anesthesia that was induced using
ethyl ether. Whole brain (-ce), cerebral cortex, midbrain, cerebel-
lum, striatum, hippocampus and liver were carefully removed
and placed on an ice-chilled petri dish for cleaning. The tissues
were weighed, cleaned with isotonic saline and kept at 20 °C till
homogenization. The handling of animals and the study protocol
were in accordance with EU Directive 2010/63/EU for animal
experiments and approved by the local Institutional Animal Care
and Use Committee (Animal Act, PD 56/13).
2.5. Behavioral testing
2.5.1. Evaluation of anxiety-like behavior
2.5.1.1. Elevated plus maze test. Elevated plus maze test was per-
formed on day 26 and during the light phase (08:00–14:00) using
two groups of mice (n = 8/group). The above task is considered to
be one of the most commonly used tests for measuring
anxiety-like behavior. The apparatus consists of two open arms
(25  5  0.5 cm) across from each other and two closed arms
(25  5  16 cm). In between there is a center platform
(5  5  0.5 cm). The open arms have a very low (0.5 cm) wall to
decrease the number of falls, whereas the closed arms have a high
(16 cm) wall to enclose the arm. The entire apparatus is 50 cm
above the floor. The two experimental groups of mice were trans-
ferred to the behavioral testing room 60 min prior to the beginning
of the procedure to habituate to the conditions of the room. Each
mouse was placed in the central platform with its head towards
a closed arm and was freely allowed to explore the apparatus for
10 min in dark. The parameter assessed for studying the
anxiety-like behavior of the mice was the ratio of time spent to
the open arms to the total time spent to the open and closed arms
(Op/Op + Cl) [24]. The above parameter and the movement of each
mouse were simulated by the computing program Phobos [25].
2.5.2. Evaluation of depression-like behavior
2.5.2.1. Forced swimming test. The forced swimming test (FST) was
performed on day 27 and during the light phase (08:00–14:00).
The apparatus used in this task consists of a transparent cylinder
filled with water (24 °C) up to 15 cm deep so that the mouse’s tail
does not come in contact with the bottom of the cylinder [26].
The two experimental groups of mice (n = 8/group) were trans-
ferred to the behavioral testing room 60 min prior to the beginning
of the procedure to habituate to the condition of the room. Each
mouse was placed in the cylinders filled with water for 6 min.
The immobility time during the last 4 min was assessed as the
measure of depression-like behavior.
2.5.3. Evaluation of cognitive activity
2.5.3.1. Step-through passive avoidance task. Step-through passive
avoidance test was performed on two consecutive days in order
to examine memory consolidation. The animals that we used
(n = 8/group) were different from those used for the other two
behavioral experiments. According to previously described [27]
and slightly modified procedures a two-compartment (white/dark,
separated by a guillotine door) passive avoidance apparatus was
used. The dark compartment of the device was equipped with a
grid floor.
Roughly, on the 26th day, the first day of the test, were placed
each mouse was placed in the illuminated compartment and left
for 100 s to habituate the apparatus (habituation trial). One hour
after the habituation trial, in the acquisition trial, each mouse
was placed in the illuminated chamber and the guillotine door
was opened. Once the animal crossed all four paws in the dark
49
chamber, a foot shock (25 V, 3 mA, 5 s) was applied. The initial
latency (IL) required to enter the dark compartment was measured
(max time allowed 120 s). The animals that did not enter the dark
chamber were eliminated from the experiment. Twenty-four hours
later, on day 27, the retention trial was performed. Each mouse
was placed in the illuminated compartment of the apparatus, the
door was opened and the step-through latency time (STL) until
the mouse entered the dark chamber was recorded (maximum
time allowed 300 s). During the latter session, no electric shock
was applied. All training and testing sessions were carried out dur-
ing the light phase (08:00–14:00).
2.6. Determination of ChE activity
Tissues were homogenized (10% w/v) in ice-cold sodium phos-
phate 30 mM, pH = 7.6 with glass-Teflon homogenizer (Thomas
Philadelphia, USA, No. B 13957). The samples were centrifuged at
15,000g, for 20 min at 4 °C to recover a salt soluble fraction (SS).
The pellet was re-dissolved with equal volume of 1% (w/v)
Triton-X 100 (in sodium phosphate 30 mM, pH = 7.6) and cen-
trifuged at 15,000g, for 20 min, at 4 °C. The supernatant contained
the detergent-soluble fraction (DS) [28]. Salt- and
detergent-soluble fractions were stored at 70 °C until use.
ChE activity was determined using Ellman’s colorimetric
method [29,30]. All determinations were carried out three times
and in triplicate. The ChE activity was expressed as lmol/min/g
tissue.
2.7. Statistical analysis
The results were analyzed by Student’s t-test analysis.
Probability values of less than 0.05 were considered statistically
significant. All statistical analyses were performed using the SPSS
statistical software version 13.0.
2.8. Pharmacophore alignment using Phase and Principal component
analysis
Three-dimensional structures for 6 compounds (3 approved
drugs (donepezil, galantamine, rivastigmine) and 3 phytochemi-
cals present in the rosemary tea (caffeic acid, carnosic acid, ros-
marinic acid)) were downloaded from the Cambridge Structural
Database (CSD) and the Protein Data Bank (donepezil CSD refer-
ence code: XETKEN, galantamine CSD reference code: JINZEM,
rivastigmine CSD reference code: MAKKEQ, caffeic acid CSD refer-
ence code: FESNOG, carnosic acid CSD reference code JUWDAG
(modificated structure), rosmarinic acid PDB code: 3QNL). The
structure of luteolin-7-O-glucuronide was sketched in Maestro
(Schrodinger Suite 2012.2). All structures were minimized using
the Polak-Ribiere conjugated gradient. Bioactive conformations
were generated using ConfGen [31]. Pharmacophoric sites were
created using Phase version 3.4 [32]. Phase provides six types of
pharmacophore features: hydrogen bond acceptor (A), hydrogen
bond donor (D), hydrophobe (H), negative ionizable (N), positive
ionizable (P), and aromatic ring (R). The program then identifies
the common characteristics of the active ligands that will explain
the ligand–receptor binding by aligning their common features.
Details regarding the Principal Component analysis on the 2D
chemical property space between the approved drugs and the
determined phytochemical in the rosemary tea can be found in
the Supporting Information.
2.9. Molecular docking studies
Docking calculations of the different AChE known drugs (done-
pezil, rivastigmine, galantamine) as well as caffeic acid, carnosic
50 A.-V. Ferlemi et al. / Chemico-Biological Interactions 237 (2015) 47–57
acid, luteolin-7-O-glucuronide and rosmarinic acid, which are pre-
sent in the herbal infusion, with the recombinant human acetyl-
cholinesterase (rhAChE) cocrystallized with donepezil in the
binding site (PDB code 4EY7 [33]; subdomain A) were performed
utilizing Schrödinger’s suite 2012.2 and GlideXP [34]. Please see
Supporting Information for the details of the method.
3. Results
3.1. Total phenolic, flavonoid and anthocyanin content of rosemary
infusion
Rosemary infusion was prepared after 5 min extraction of rose-
mary leaves in hot boiled water. After lyophilization, the remaining
dry weight was 0.033 g/100 mL beverage. The total polyphenol con-
tent in the R. officinalis tea was 12.700 ± 0.008 mg GAE/100 mL,
whereas the content of the tea in flavonoids and monomeric antho-
cyanins was 1.444 ± 0.069 mg QE/100 mL and 0.619 ± 0.038 mg
3-cyanidin glucoside equivalents/100 mL, respectively.
3.2. LS–MS based exploration of the phytochemical profile of the
rosemary extract
The LC/DAD/ESI-MSn analysis of the rosemary extract led to the
separation and identification of the majority of the constituents;
overall, 16 compounds were identified and those belong to three
representative classes of constituents: diterpenes, flavonoids and
hydroxycinnamic derivatives. Because polyphenols contain one
or more hydroxyl and/or carboxylic acid groups, MS data were
acquired in negative ionization mode. Identification of the com-
pounds was carried out by comparing retention times and masses
with those of the 4 authentic standards (quinic acid, rosmarinic
acid, carnosol and carnosic acid). For the remaining 12 compounds
for which no standards were available, identification was based on
accurate mass measurements of the pseudomolecular [M H] ions
and their fragmentation pattern, as has been documented in the
literature. The total ion current (TIC) chromatogram is presented
in Fig. 2A while the UV chromatogram at 284 nm is presented in
Fig. 2B. Data obtained from the ESI-MSn analysis of the extracts
are summarized in Table 1. For a detailed analysis of the mass
spectrometry data and the associated compounds please see
Supporting Information.
3.3. Quantitative determination of the major phenolic compounds
The major phenolic compounds (indicated 3, 6 and 7 in
Fig. 2A and B) were quantified by means of an HPLC/diode array
method. Since reference compounds were not available for most
Fig. 1. Effect of rosemary tea consumption on mice behavior: [A] initial latency (IL) and Step-through latency (STL) of adult male mice in both control and Rosemary-treated
group, in the step-through test. No significant difference was observed between the Ro-treated and the control group. [B] The ratio of time spent to the open arms to the total
time to the open and closed arms (Op/Op + Cl). [C] Evaluation of the immobility time. Each column represents Mean ± S.E. (n = 8). Statistical analysis was performed by
Students t-test. ⁄Statistical difference between control and Rosmarinus-treated mice group (p < 0.05). Each column represents Mean ± S.E. (n = 8).
of the detected compounds, caffeic acid derivative was quantitated
as caffeic acid and luteolin-glucuronide was quantitated as
luteolin-7-O-glucoside. The calibration curves were proved to be
linear in the ranges 10–80 mg/L with R2 0.988 (rosmarinic acid),
2–20 mg/L with R2 0.994 (caffeic acid) and 2–20 mg/L with R2
0.991 (luteolin-7-O-glucoside). The concentration of rosmarinic
acid was found 70.78 ± 5.15 mg/g dry weight of freeze dried rose-
mary infusion (2.33 mg/100 mL of the infusion), of caffeic acid
derivative 8.48 ± 0.21 mg/g dry weight (0.28 mg/100 mL of the
infusion) and of luteolin-glucuronide 4.44 ± 0.43 mg/g dry weight
(0.15 mg/100 mL of the infusion). Carnosol, quinic acid and carno-
sic acid were detected in trace amounts.
3.4. Behavioral effects of rosemary infusion consumption in mice
The regular recording of food and liquid consumption revealed
that the animals did not show preference to the rosemary tea and
that its daily drinking (15 mL) did not affect the consumption of
food. Moreover, we tested the changes on the body weight and on
the weight of cerebral and hepatic tissues of mouse and we found
no significant differences between the two groups. Macroscopic
examination of the tissues is in line with the above observation.
Step-through passive avoidance test was used in order to deter-
mine the learning/memory status of the rodents after the treat-
ment period. The results are shown in Fig. 1A. Initial Latency
time (IL), which is relative to the motor and explorative activity
of mice, showed no statistically significant differences (p > 0.05)
between groups, demonstrating that controls and treated mice
behave the same during the acquisition trial. Step-through
Latency time (STL), which was recorded during the retention trial,
shows the consolidation of the reinforced stimuli in the memory of
rodents. No significant difference was observed between the
Rosemary-treated and the control group. Apparently, R. officinalis
leaf tea 4-weeks consumption had no effect on learning ability of
normal mice.
Elevated plus maze test assessed the anxiety-like behavior of
the rodents after the treatment period. The results are listed in
Fig. 1B. Time spent in open and closed arms was recorded, and
the parameter op/op + cl (time in open arms/time in both open
and closed arms) was calculated. Between the two experimental
groups there was statistically significant difference (p < 0.05). The
results demonstrated that rosemary leaf tea had an
anxiolytic-like effect on mice.
The last behavioral test was the forced swimming test. The
results are listed in Fig. 1C. Between the two experimental groups,
the immobility time (IT) was significantly (p < 0.05) decreased in
Rosemary-treated group of mice (55.71%) as they appeared to
struggle more in the water trying to find a way out of it. The results
 A.-V. Ferlemi et al. / Chemico-Biological Interactions 237 (2015) 47–57 51

Fig. 2. Total ion chromatogram (TIC) of aqueous rosemary extract [A] UV chromatogram at 284 nm. 1: caffeic acid-O-hexoside derivative, 2: quinic acid, 3: caffeic acid
derivative, 4: isorhamnentin-3-O-hexoside, 5: homaplantaginin, 6: luteolin-7-O-glucuronide, scutellarein or isoscutellarein, 7: rosmarinic acid, 8: luteolin-acetyl-
glucuronide, 9: cirsimaritin, 10: rosmanol isomer, 11: rosmanol isomer, 12: rosmanol isomer, 13: rosmadial, 14: notohamosin R, 15: carnosol, 16: carnosic acid, 17: unknown
[B].

Table 1
Peak assignments of rosemary extract (Rt: retention time).

P Rt [M H] (m/z) (%) –MS2 [M H] (m/z) (%) –MS3 [base peak]- (m/z) (%) Compounds
(min)
1 3.6 455 (100), 387 (72) (387) 341 (100), (455) 409 (100), 341 (387) 179, 161, (455) 341, Caffeic acid-O-hexoside derivative
(40) 363
2 3.7 191 (100), 383 (96) (383) 191 (100), (191) 127 (86), 147 (383) 170, 173 Quinic acid
(100)
3 4.9 459 (100) 161 (100), 179 (34) 158 (100) Caffeic acid derivative
4 9.6 477 (100) 315 (100), 300 (37) 300 (100), 271 (41) Isorhamnetin-3-O-hexoside
5 11.1 461 (100) 299 (100), 284 (70) 284 (100), 297 (97) Homaplantaginin
6 11.7 461 (100), 777 (34) 285 (100) 285, 217, 241 Luteolin-7-O-glucuronide, scutellarein or
isoscutellarein
7 12.5 359 (100), 473 (31), 719 161 (100), 197 (36), 178 (35) 133 (100), 117 Rosmarinic acid
(27)
8 14.0 503 (100) 285 (100), 443 (48) 283, 241 Luteolin-acetyl-glucuronide
9 21.3 313 (100) 298 (100), 311 (32) 283, 297 Cirsimaritin
10 22.6 459 (100), 345 (36) 345 (100), 301 (14) 301, 283 Rosmanol isomer
11 23.5 345 (100), 459 (69) 283 (100) (345) 268, 239, 281 Rosmanol isomer
12 24.1 345 (100), 283 (56) 283 (100), 345 (75) 265, 281 Rosmanol isomer
13 28.6 343 (100) 343 (100), 299 (78) – Rosmadial
14 29.1 457 (100), 299 (18) 299 (100), 343 (23) – Notohamosin R
15 29.8 329 (100), 443 (77) 286 (100) 285, 270 Carnosol
16 33.2 331 (100) 287 (100) 244, 285, 269, 217 Carnosic acid
17 34.4 265 (13) 265, 169 113 Unknown

in this case demonstrated the anti-depressant-like effect on mice
of rosemary leaf tea.
3.5. Effect of rosemary infusion on ChE activity in whole brain,
individual brain areas and liver
AChE activity constitutes the major fraction of total ChE activity
in whole brain (-ce) and cerebellum, whereas BChE activity pre-
dominates in liver. In order to evaluate the possible role of ChE,
we studied the effect on two fractions of the enzyme: the
salt-soluble (SS) and the detergent-soluble (DS) fraction. In the
SS fraction the cytosolic G1 isoform of the AChE predominates,
whereas the DS fraction contains mainly the membrane-bound
G4 isoform of the enzyme. G1 and G4 isoforms of BChE are evenly
distributed in between soluble and membrane-bound species [35],
thus none of them predominates in the fractions.
The results concerning the whole brain (-ce), brain areas and
the liver ChE activity are described in Table 2. Firstly, the activity
of the DS fraction is much higher than the activity of the SS fraction
in brain tissues relatively to the liver. In whole brain samples, rose-
mary tea consumption resulted in a significant inhibition of ChE
activity in both SS and DS fraction (19.29% and 27.72%) relatively
to the control group.
As far as brain areas are concerned, the results demonstrated
that ChE activity was significantly inhibited in both SS and DS frac-
tion except of hippocampus, where the activity of SS-ChE was
insusceptible. The results are shown in Table 3. In detail, in cere-
bral cortex the inhibition of ChE activity in SS and DS fraction
was 29.93% and 30.83%, respectively. In midbrain we observed that
52 A.-V. Ferlemi et al. / Chemico-Biological Interactions 237 (2015) 47–57

Table 2
Effect of rosemary tea consumption (2% w/v) on ChE levels of mice tissues.

ChE (lmol/min/g tissue)

Tissues Homogenates fractions Control mice Rosemary-treated mice Percentage of decrease (%)
Whole brain SS 10.03 ± 0.75 8.09 ± 0.40⁄ 19.29
DS 82.38 ± 4.93 46.33 ± 0.99⁄ 27.72
Liver SS 44.43 ± 0.94 23.25 ± 2.14⁄ 44.77
DS 15.12 ± 0.71 8.93 ± 0.87⁄ 15.13

(a) Data are Mean ± S.E. (n = 8). Statistical analysis was performed by Student’s-t-test.
(b) ⁄Statistical difference between Control and Rosemary-treated mice (p < 0.05).
(c) Percentage decrease (;) of ChE values compared to the control group.

Table 3
Effect of rosemary tea consumption (2% w/v) on ChE levels of mice brain areas.

ChE (lmol/min/g tissue)

Tissues Homogenates fractions Control mice Rosemary-treated mice Percentage of decrease (%)
Cerebral cortex SS 7.73 ± 0.75 5.42 ± 0.64⁄ 29.93
DS 32.49 ± 1.98 22.47 ± 2.18⁄ 30.83
Midbrain SS 16.70 ± 0.90 14.07 ± 1.70⁄ 21.31
DS 74.52 ± 2.51 50.48 ± 4.72⁄ 32.25
Cerebellum SS 8.58 ± 0.66 5.89 ± 0.38⁄ 31.30
DS 16.43 ± 0.58 10.53 ± 1.00⁄ 35.92
Striatum SS 8.02 ± 0.55 6.46 ± 0.46⁄ 24.56
DS 73.52 ± 6.63 43.61 ± 3.90⁄ 39.59 %
Hippocampus SS 8.64 ± 0.67 8.15 ± 0.56
DS 38.72 ± 2.28 24.45 ± 1.90⁄ 34.22

(a) Data are Mean ± S.E. (n = 8). Statistical analysis was performed by Student’s t-test.
(b) ⁄Statistical difference between Control and Rosemary-treated mice (p < 0.05).
(c) Percentage decrease (;) of ChE values compared to the control group.

ChE activity was inhibited by 21.31% in SS fraction and 32.25% in
DS fraction. Moreover, in cerebellum the inhibition was 31.30%
and 35.92%, respectively in SS and DS fraction. In striatum, the
inhibition was significant in both SS (24.56%) and DS fraction
(39.59%). In hippocampus rosemary leaf tea did not affect
SS-fraction activity. However, ChE’s activity in DS fraction was
inhibited by 34.22%.
In the case of liver, rosemary leaf tea led to the statistically sig-
nificant inhibition (p < 0.05) of ChE activity in both fractions, as it is
demonstrated in Table 2. In detail, ChE inhibition in SS and DS frac-
tion was 44.77% and 15.13%, respectively.
3.6. Pharmacophore alignment of the phenolic compounds with drugs
established for their anti-acetylcholinesterasic activity – molecular
docking calculations in the AChE site
The anti-acetylcholinesterasic activity of rosemary tea
prompted us to explore a potential pharmacophore similarity of
the determined phytochemicals with approved drugs conferring
anti-acetylcholinesterasic activity. The structural alignment of
Donepezil with rosmarinic acid and luteolin-7-O-glucuronide
(Fig. 3(B1)) shows 5 common pharmacophore sites: 2 aromatic
rings (brown features) at a distance of 6.437 Å, and three hydrogen
bond acceptors (red spheres). Four common features are found in
the alignment of Rivastigmine and carnosic acid (Fig. 3(B2)): 2
hydrogen bond acceptors, 1 aromatic ring and 1 hydrophobic site
(green sphere). Since both molecules are rigid (only 4 bioactive
conformations are found by ConfGen software) the fact that their
alignment finds 4 common pharmacophore sites suggests that
both could present similar/common ligand binding interaction
profile. The superposition of Galantamine (that has only two
bioactive conformations) with rosmarinic acid and luteolin-7-O-
glucuronide (Fig. 3(B3)) showed 4 common pharmacophore sites:
1 aromatic ring and 3 hydrogen bond acceptors. These compar-
isons illustrated a considerable structure similarity between phy-
tochemicals present in the rosemary tea and known drugs
presenting anti-acetylcholinesterasic activity. A potential pharma-
cophore overlay between the approved drugs and the rosemary tea
phytochemicals could be identified suggesting the determined
common function – anti-acetylcholinesterastic activity of these
molecules. This pharmacophore similarity was further exploited
through docking calculations of caffeic acid to the X-ray structure
of AChE. The three dimensional plots, highlighting the interactions
developed between major phytochemicals in the herbal infusion
(rosmarinic acid, caffeic acid and luteolin-7-O-glucuronide) along
with carnosic acid, one of the main constituents of rosemary leaves
found in traces in rosemary infusion because of its low solubility in
water, and AChE are illustrated in Fig. 4, whereas the relevant 2D
plots are presented in Fig. S7. From a direct comparison of these
results to the relevant data obtained from the docking calculations
of the AChE known drugs (Donepezil, Galantamine, Rivastigmine,)
to AChE (Figs. S4–S6) it is further strengthened that the common
pharmacophore groups, pinpointed above, can be utilized to
develop similar interactions to the active site of AChE. The relevant
interactions are presented in a tabulated format in Table S1. The
four compounds show favorable binding scores (Table S1) that
are in close agreement with the docking scores of the approved
drugs as calculated using GlideXP. In addition, the four compounds
interact with the same residues that donepezil and galantamine
interact with, as shown in the 3D crystal structures of these com-
pounds with AChE (Figs. S5 and S6). The superposition of the best
binding pose of luteolin-7-O-glucuronide with the crystal structure
 A.-V. Ferlemi et al. / Chemico-Biological Interactions 237 (2015) 47–57 53

Fig. 3. (A) Results of the principal components analysis comparing in the chemical property space the determined phytochemicals and approved drugs with known anti-AChE
activity. (B) The pharmacophore alignments of: (1) donepezil (blue) with rosmarinic acid (green) and luteolin-7-O-glucuronide (purple). (2) rivastigmine (green) and carnosic
acid (blue). (3) galantamine (blue) with rosmarinic acid (green) and luteolin-7-O-glucuronide (purple) (4) rosmarinic acid (blue) with caffeic acid (green) and luteolin-7-O-
glucuronide (purple). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of donepezil bound to human acetylcholinesterase show remark-
able similarities, further validating our pharmacophore approach
(Fig. S8).
4. Discussion
The therapeutic application of R. officinalis in folk medicine in
the management of mood disorders is established. Several reports
have shown its ethnopharmacological uses for the treatment of
physical and mental fatigue, improvement of memory and treat-
ment of nervous agitation, hysteria and depression, among other
applications [6]. However, research has focused on rosemary
essential oil, alcoholic and aqueous extracts. It is the first time that
R. officinalis leaf tea was studied for its composition and possible
positive effects on mood regulation and ChE activity.
The determination of total phenolic content showed that the
amount of rosemary tea polyphenols is similar to the content that
we have recorded for other Labiatae species of Greek flora, such as
several Sideritis clandestina subsp. [36]. LC–MS analysis of rosemary
tea showed the presence of 16 compounds that are classified into
the categories of flavonoids, diterpenes and hydroxycinnamic
derivatives; it was shown that rosmarinic acid (RA) was the major
bioactive compound of the infusion, followed by a caffeic acid
derivative and luteolin 7-O-glucuronide. RA was, also, the major
component of the water-soluble extract of rosemary leaf in the
study of del Bano et al. [37], but it was found only in trace amounts
in methanol and acetone extracts.
Regular recording of mouse body weight and liver weighing
right after its removal, revealed that there were no differences
between the treated and the control group. Furthermore, macro-
scopic examination revealed that liver tissues of both groups were
normal in appearance, color and shape indicating that the pro-
longed consumption of 130 mg/kg body weight of rosemary tea
did not affect mouse liver. Our results are reinforced by Daher
and Kashour [23] who reported that administration to rats of
54 A.-V. Ferlemi et al. / Chemico-Biological Interactions 237 (2015) 47–57
different concentrations of rosemary extract (100, 200, 500 mg/kg
body weight) via drinking water for one month resulted in no liver
toxicity. It should be mentioned, though, that the chosen treatment
allowed us to examine the positive effects of oral administration of
rosemary tea without toxicity effects, however it is such a high
concentration that could not be achieved by nutritional interven-
tion but only with supplementation.
Behavioral study revealed that rosemary’s daily oral adminis-
tration to adult male mice reduced significantly both the
anxiety- and the depression-like behavior. However, even though
rosemary is traditionally considered as a cognitive enhancer [5],
we found that the consumption of the infusion had
non-significant effect (though positive) on memory/learning abil-
ity of mice. Several studies have shown the protective effects of
rosemary alcoholic/hydroalcoholic extracts or of main components
of its leaves in mood regulation through several neurotransmitter
systems [38,39]. One of the main components of rosemary tea,
RA, has been thoroughly investigated for its anti-depressant and
anxiolytic actions. An in vivo study demonstrated that when RA
was intraperitoneally administered to adult male mice at a dose
of 2 mg/kg, it reduced significantly the immobility time during
the FST [40]. The same applies to caffeic acid (CA) as it also
presented antidepressant effects. Moreover, when a variety of
doses of RA was administrated i.p. in adult male mice, a
dose-dependent anxiolytic action of RA was observed [41].
The neurobiological background of both anxiety and depression
is under investigation. In these severe mood deregulations cholin-
ergic system fulfils a rather crucial role. In our study, both SS- and
DS-ChE activity was inhibited significantly in all examined brain
regions and liver, apart from hippocampus SS-ChE activity (no
change). Hippocampus is known for its important involvement in
memory process and mood regulation, i.e. anxiety-like behaviors
and depression [42]. However, the exact way by which this brain
region contributes to behavior needs to be clarified. A correlation
between the unchanged hippocampal SS-ChE activity and mem-
ory/learning could not be hypothesized since we have demon-
strated that the two parameters are not correlated [43]. The
non-correlation of the two parameters was reinforced by
Machado et al. [44] who showed that a hydroalcoholic extract of
R. officinalis decreased ChE hippocampal activity without altering
learning deficit. Moreover, they also demonstrated that the
rosemary extract which contained rosmarinic acid, carnosol,
betulinic acid, oleanolic acid, and ursolic acid exerted an
antidepressant-like effect in bulbectomized mice.
In order to get some insight to the way with which R. officinalis
tea exerts the observed properties of our study, the network of
interactions among the different bioactive compounds and cellular
constituents needs to be characterized [45]. RA, for example, when
administrated intraperitoneally in rats in a variety of doses,
reduced significantly AChE levels in individual brain regions, such
as frontal cortex and hippocampus [46]. Moreover, caffeic acid was
administered to adult male rats once a day for 30 days at the doses
of 10, 50 and 100 mg/kg by gavage [47] and the results demon-
strated that AChE activity was decreased significantly in cerebral
cortex and striatum compared to the control group of rats,
whereas, the same doses of caffeic acid provoked a significant
increase in AChE activity in hippocampus and pons. In addition,
Costa et al. [48] demonstrated that water/ethanol extracts of wild
and cultured plants of Lavandula viridis, which accumulate high
amounts of caffeoyliquinic acids, rosmarinic acid and luteolin, pos-
sess significant anti-acetylcholinesterasic activity.
Since, AChE activity predominates in cerebral tissues compared
to BChE [49], where it plays significant role in neurotransmition,
one could wonder about the effectiveness of plant compounds on
the enzyme of AChE. First, it should be clarified whether phenolics
can cross blood–brain barrier and reach different cerebral regions.
Indeed, pharmacokinetic studies have shown after i.p. administra-
tion of RA (60 mg/kg) to rats that the compound was found at very
low concentration in plasma (AUC0–6h 6.6 mg h/L and T1/2 0.75 h),
probably due to liver metabolism, while according to the AUC
low level of RA in brain, this phenolic compound does not effi-
ciently cross the blood–brain barrier [50]. Baba et al. [51] showed
that RA orally administrated in rats (50 mg/kg) was absorbed and
metabolized as conjugated and/or methylated forms and that the
majority of RA absorbed was degraded into conjugated and/or
methylated forms of caffeic acid, ferrulic acid and m-coumaric acid.
These metabolites could be further metabolized according to
Vaquero et al. [52]; LC–MS/MS and pharmacokinetic analysis of
the main metabolites of a rosemary extract that was orally admin-
istered to Zucker rats revealed the presence of CA (1), CA glu-
curonide (2), CA 12-methyl ether (3), hydroxyrosmariquinone
(caffeic acid derivative – 4), as well as carnosol and its glucuronide
in plasma. Finally, the authors reported that CA and carnosol were
found in significant amounts in brain, whereas compounds 2, 3, 4
were also detected in traces. Carnosic acid is, also, accumulated
in mouse brain in in vivo and in vitro experiments as well, where
it accumulates in high concentrations in neurons and in low con-
centrations in non-neuronal cells and exerts several protective
antioxidant effects as shown by Satoh et al. [53]. Finally, it is estab-
lished that polyphenols cross blood–brain barrier [54,55] and it is
plausible that they accumulate in several brain regions at different
degrees [56]. These observations about the metabolism of rose-
mary extract and/or its main components, i.e. RA, CA, carnosic acid
and polyphenols, support the potential benefits against brain dis-
orders derived from the consumption of rosemary.
Secondly, a challenging goal is the investigation whether plant
specific phytochemicals affect directly AChE enzyme with respect
to established drugs such as donepezil and rivastigmine. It has
been demonstrated that caffeic acid inhibits in vitro both AChE
and butyrylcholinesterase (BChE) activities in dose-dependent
manner [57]. Moreover, Szwajgier [58] tested several phenolic
acids found in beer and concluded that ferulic, p-coumaric and caf-
feic acids were the most potent inhibitors against cholinesterases,
indicating that the type of acid, cinnamic or benzoic, as well as the
presence of OH-group in para- or meta-position in the molecule
elevated its inhibitory activity. Conforti et al. [59] showed that caf-
feic acid acts as an efficient AChE inhibitor with IC50 value
32.4 lg/mL, while luteolin had the most promising anti-AchE
activity (IC50: 25.2 lg/mL). As far as luteolin is concerned,
Dhananjayan et al. [60] in a molecular docking and in vitro acetyl-
cholinesterase inhibition study, indicated that this particular
polyphenol showed the lowest IC50 (135 lg/mL) value among
other flavonoids and terpenoids, while tacrine that was used as ref-
erence had only 0.5-fold lower IC50 (92 lg/mL). However, molecu-
lar docking revealed that luteolin showed the lowest binding
energy to the enzyme of AChE among other flavonoids. In the study
of Szwajgier [61] the anti-cholinesterase activity of several pheno-
lic compounds was tested and carnosic acid possessed the highest
inhibitory activity, followed by rosmarinic and caffeic acid. In
another study, rosmarinic acid showed almost 50% inhibition of
AChE in the concentration of 0.25 mg/mL and caffeic acid was
almost, equally effective in the high concentrations but in low con-
centration its inhibitory activity was far below 50% [62].
Taking account the phytochemical composition of rosemary tea,
literature references which report the penetration of these com-
pounds in brain, their in vitro AChE inhibitory activity and the
few in silico studies about the binding of phenolic compounds on
AChE, we hypothesized that some of our main components may
act as common drug inhibitors against the enzyme, thus we exam-
ined the possible common features of these compounds with
known AChE inhibitors. We selected the caffeic, rosmarinic and
carnosic acid, while luteolin-7-O-glucuronide was chosen among
 A.-V. Ferlemi et al. / Chemico-Biological Interactions 237 (2015) 47–57
Fig. 4. The binding pose of: (A) rosmarinic acid (green) in the active site of AChE, (B) luteolin-7-O-glucuronide (green), (C) caffeic acid (green) and (D) carnosic acid (green).
The important residues are shown in purple. Hydrogen bonds are shown in yellow dashed lines. Numbering of residues from rhAChE (pdb codes 3EY6 and 3EY7) is used
unless explicitly noted. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the few determined polyphenols. Indeed, the principal component
analysis of the chemical property space sampled by the rosemary
tea phytochemicals and the approved drugs (conferring
anti-acetylcholinesterasic activities) illustrated that phytochemi-
cals cover a larger part of the relevant chemical space (Fig. 3A).
To get more insight to the common anti-acetylcholinesterasic
activities presented among the rosemary tea phytochemicals and
approved drugs we exploited potential common pharmacophore
features in the 3D space. We therefore utilized an established com-
putational approach based on pharmacophore alignment and we
found that the phytochemicals adopted a large number of common
sites with drugs suggesting that they develop a similar interaction
profile with the drug protein targets. To further exploit the identi-
fied pharmacophore similarity we performed docking calculations
of the different compounds to the X-ray structure of AChE. These
results strengthened the fact that the common pharmacophore
groups, reported above, may function in similar way as AChE
known inhibitors via developing alike interactions to the active site
of AChE.
With regard to the binding type of the isolated compounds on
AChE molecule, it is known that in order to achieve covalent mod-
ification of a protein target a highly electrophilic species (e.g.,
a-halo ketone, a,b-unsaturated ketone, fluorophosphonate (FP) or
cyanamide) needs to be incorporated into the inhibitor. The pres-
ence of Michael acceptors in an inhibitor scaffold also has been
used to covalently inactivate cysteine residues in their target
enzymes. Even though a 1,2 addition could happen to rosmarinic
acid, docking calculations into the binding site of AChE indicates
that the pose taken by rosmarinic acid does not favor a nucle-
ophilic attack from Ser203 (Fig. 4A). Furthermore the study by
Marcelo et al. [63] also suggests that rosmarinic acid does not inhi-
bit AChE through covalent binding. The findings of Anwar et al.
[47] , Oboh et al. [57] and Khan et al. [64] revealed that caffeic acid
inhibited AChE activities in dose-dependent manner thus indicat-
ing non-covalent inhibition.
Conclusively, the marked antidepressant/anxiolytic-like and
anti-cholinesterasic effects of R. officinalis infusion support the
beneficial role of regular drinking of rosemary. Molecular modeling
study indicated that some of the main rosemary infusion com-
pounds may function against AChE molecule as known AChE inhi-
bitors, explaining partially the observed anti-acetylcholinesterasic
55
effects of our tea. However, herbal extracts are chemically complex
mixtures containing multiple major and minor constituents with
multiple potential targets and mechanisms, and thus further work
is necessary to elucidate the mechanisms although the behavioral
effects have been conclusively demonstrated in many studies.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Transparency Document
The Transparency document associated with this article can be
found in the online version.
Acknowledgments
This research project was partially supported by BIOFLORA, net-
work of University of Patras and has been co-financed by the
European Union (European Regional Development Fund – ERDF)
and Greek national funds through the Operational Program
‘‘THESSALY-MAINLAND GREECE AND EPIRUS-2007–2013’’ of the
National Strategic Reference Framework (NSRF 2007–2013).
Special thanks are given to the Mass Spectrometry Unit of
University of Ioannina for providing access to LC–MS/MS facilities.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cbi.2015.04.013.
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