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Dracocephalum moldavica attenuates scopolamine-induced cognitive impairment through activation of hippocampal ERK-CREB signaling in mice
Journal Pre-proof Dracocephalum moldavica attenuates scopolamine-induced cognitive impairment through activation of hippocampal ERK-CREB signaling in mice Ponnuvel Deepa, Ho Jung Bae, Hyeon-Bae Park, So-Yeon Kim, Songmun Kim, Ji Woong Choi, Dong Hyun Kim, Xiang-Qian Liu, Jong Hoon Ryu, Se Jin Park PII:
S0378-8741(19)31446-1
DOI:
https://doi.org/10.1016/j.jep.2020.112651
Reference:
JEP 112651
To appear in:
Journal of Ethnopharmacology
Received Date: 10 April 2019 Revised Date:
3 January 2020
Accepted Date: 2 February 2020
Please cite this article as: Deepa, P., Bae, H.J., Park, H.-B., Kim, S.-Y., Kim, S., Choi, J.W., Kim, D.H., Liu, X.-Q., Ryu, J.H., Park, S.J., Dracocephalum moldavica attenuates scopolamine-induced cognitive impairment through activation of hippocampal ERK-CREB signaling in mice, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/j.jep.2020.112651. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Graphical abstract
1
Dracocephalum moldavica attenuates scopolamine-induced cognitive impairment
2
through activation of hippocampal ERK-CREB signaling in mice
3 4
Ponnuvel Deepa1, Ho Jung Bae2, Hyeon-Bae Park1, So-Yeon Kim1, Songmun Kim1, Ji
5
Woong Choi5, Dong Hyun Kim6, Xiang-Qian Liu4, Jong Hoon Ryu2, 3, *, Se Jin Park1, *
6 7
1
8
Chuncheon, Republic of Korea
9
2
School of Natural Resources and Environmental Sciences, Kangwon National University,
Department of Life and Nanopharmaceutical Sciences and
3
Department of Oriental
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Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, Republic of
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Korea
12
4
13
5
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Pharmaceutical Sciences, Gachon University, Incheon, Republic of Korea
15
6
16
Convergence Bio-Health, Dong-A University, Busan, Republic of Korea
School of Pharmacy, Hunan University of Chinese Medicine, Changsha, China Laboratory of Neuropharmacology, College of Pharmacy and Gachon Institute of
Department of Medicinal Biotechnology, College of Health Sciences and Institute of
17 18
*
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Se Jin Park at School of Natural Resources and Environmental Sciences, Kangwon National
20
University, Chuncheon, Republic of Korea; [email protected]
21
J.H. Ryu at Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung
22
Hee University, Seoul, Republic of Korea; [email protected]
Corresponding Authors
23 24
1
25
Authors E-mail:
26
[email protected] (P. Deepa); [email protected] (H.J. Bae)
27
[email protected] (Hyun-Bae Park); [email protected] (So-Yeon Kim)
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[email protected] (Songmun Kim); [email protected] (Xiang-Qian Liu)
29
[email protected] (Dong Hyun Kim); [email protected] (Ji Woong Choi)
30
[email protected] (Jong Hoon Ryu); [email protected] (Se Jin Park)
31 32
Running title: Memory-ameliorating effect of Dracocephalum moldavica
2
33
List of abbreviations
34
AChE, acetylcholinesterase
35
Aβ, amyloid beta
36
AD, Alzheimer’s disease
37
BDNF, brain-derived neurotrophic factor
38
CaMKII, Ca2+/calmodulin-dependent protein kinase II
39
CNS, central nervous system
40
CREB, cAMP response element-binding protein
41
DNZ, donepezil
42
EEDM, ethanolic extract of Dracocephalum moldavica
43
ERK, extracellular signal regulated kinase
44
PKA, protein kinase A
45
3
46
Abstract
47
Ethnopharmacological relevance: Dracocephalum moldavica (Moldavian balm) has been
48
traditionally used for the treatment of intellectual disabilities, migraines and cardiovascular
49
problems in East Asia. Recent scientific studies have demonstrated the usefulness of this
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plant to treat neurodegenerative disorders, including Alzheimer’s disease.
51
Aim of the study: This study aimed to investigate the effects of the ethanolic extract of D.
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moldavica leaves (EEDM) on scopolamine-induced cognitive impairment in mice and the
53
underlying mechanisms of action.
54
Materials and methods: The behavioral effects of EEDM were examined using the step-through
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passive avoidance and Morris water maze tasks. To elucidate the underlying mechanism, we
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tested whether EEDM affects acetylcholinesterase activity and the expression of memory-
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related signaling molecules including extracellular signal-regulated kinase (ERK) and cAMP
58
response element-binding protein (CREB) in the hippocampus.
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Results: EEDM (25, 50 or 100 mg/kg) significantly ameliorated the scopolamine-induced
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step-through latency reduction in the passive avoidance task in mice. In the Morris water
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maze task, EEDM (50 mg/kg) significantly attenuated scopolamine-induced memory
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impairment. Furthermore, the administration of EEDM increased the phosphorylation levels
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of ERK and CREB in the hippocampus but did not alter acetylcholinesterase activity.
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Conclusions: These findings suggest that EEDM significantly attenuates scopolamine-
65
induced memory impairment in mice and may be a promising therapeutic agent for
66
improving memory impairment.
67 68
Keywords: Dracocephalum moldavica; memory impairment; Alzheimer’s disease;
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scopolamine; extracellular signal regulated kinase; cAMP response element-binding protein
70 71
4
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1. Introduction
73
Alzheimer’s disease (AD) is mainly characterized by memory deficits and mental
74
dysfunction; the former is known to be mainly correlated with declines in cholinergic
75
neurotransmission systems (Francis et al., 1999). Behavioral studies have shown that anti-
76
cholinergic drugs impair cognitive function in healthy humans and animals (Atri et al., 2004;
77
Flood and Cherkin, 1986). Accordingly, blockade of the cholinergic system with muscarinic
78
cholinergic receptor antagonists (e.g. scopolamine) is widely used to induce cognitive
79
impairment (Klinkenberg and Blokland, 2010). Moreover, several synthetic drugs, including
80
cholinesterase inhibitors, have been used for cognitive enhancement. Although synthetic
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memory-enhancing drugs effectively improve memory performance, they have several
82
adverse effects, such as nausea, vomiting, diarrhea and anorexia (Gauthier, 2001). Thus,
83
many studies have focused on the identification of novel drugs, particularly herbal plants, to
84
treat various neurodegenerative diseases, including AD.
85
Dracocephalum moldavica L. (Lamiaceae, Labiatae) is a perennial aromatic herb
86
native to central Asia, northern China, and eastern and central Europe, and is commonly
87
referred to as Moldavian balm. Because it is naturally warm and fragrant, D. moldavica can
88
affect the central nervous system (CNS), cardiac tissues, and blood circulation (Liu et al.,
89
2018). Accordingly, D. moldavica has been traditionally used for the treatment of heart
90
disease, blood pressure, angina, atherosclerosis, neuralgia, migraines, headaches and
91
toothaches (Dastmalchi et al., 2007; Liu et al., 2018; Maimaitiyiming et al., 2014; Zhao et al.,
92
2017). Additionally, recent studies have also confirmed that D. moldavica has various
93
pharmacological effects on the CNS, such as neuroprotection against rat cerebral ischemia
94
reperfusion injury (Jia et al., 2017; Zeng et al., 2018), anti-oxidative and anti-inflammatory
95
properties in an animal model of AD (Liu et al., 2018) and the promotion of prolonged
96
pentobarbital-induced sleeping time and sedation in mice (Martinez-Vazquez et al., 2012). 5
97
Furthermore, phytochemical studies have revealed that D. moldavica primarily contains
98
rosmarinic acid, oleanolic acid, chlorogenic acid, ferulic acid, caffeic acid, p-coumaric acid,
99
apigenin, quercetin, acacetin, tilianin and luteolin (Li et al., 2016). We and several groups
100
have reported that scopolamine-induced cognitive impairment is ameliorated by oleanolic
101
acid (Jeon et al., 2017), rosmarinic acid (Qu et al., 2017) and chlorogenic acid (Kwon et al.,
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2010) which are documented constituents of D. moldavica.
103
Extracellular signal-regulated kinase (ERK) and cAMP response element-binding
104
protein (CREB) signaling molecules are known to be involved in cognitive functions. ERK
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belongs to the mitogen-activated protein kinase family member and activates CREB, which
106
regulates cellular processes for the regulation of long-term synaptic plasticity and the
107
stabilization of new memories (Adams and Sweatt, 2002; Kelleher et al., 2004). Multiple
108
studies have confirmed that improvements in cognitive abilities are facilitated by the
109
activation of ERK signaling (Ciccarelli and Giustetto, 2014; Kim et al., 2012). CREB is a
110
transcription factor that binds to the promoter regions of many neuronal genes associated
111
with learning, memory and synaptic plasticity (Alberini, 2009). Thus, the activation of the
112
ERK-CREB signaling cascade is necessary for the formation and storage of memories in the
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hippocampus. It should be noted that a total flavonoid extract of D. moldavica has been
114
reported to attenuate β-amyloid-induced neurotoxicity through the activation of neurotrophic
115
pathways, including the ERK-CREB-brain-derived neurotrophic factor (BDNF) pathway (Liu
116
et al., 2018).
117
Based on previous studies, we hypothesized that D. moldavica may cure cognitive
118
disorders by targeting ERK/CREB signaling. However, no reports have described the
119
memory-ameliorating effect of D. moldavica on cognitive impairments due to cholinergic
120
blockade. Hence, the aim of this study was to investigate whether the ethanolic extract of D.
121
moldavica (EEDM) attenuates the scopolamine-induced cognitive impairment in mice using 6
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the passive avoidance and Morris water maze tasks. We also investigated whether EEDM
123
affects the phosphorylation levels of ERK and CREB in the hippocampus.
124 125
2. Materials and methods
126
2.1. Animals
127
Male CD1 ICR mice (6 weeks old, 25–30 g) were purchased from the Orient Co. Ltd.,
128
a branch of Charles River Laboratories (Seoul, Korea). The mice were housed in groups of 5
129
per cage, provided with ad libitum access to food and water, and kept under a 12 h light/dark
130
cycle (lights on 07:00–19:00) at a constant temperature (23 ± 1 ºC) and relative humidity (60
131
± 10%). Animal treatment and maintenance were carried out in accordance with the
132
Principles of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and with
133
the Animal Care and Use Guidelines issued by Kyung Hee University, Republic of Korea
134
(approval number: KHUASP(SE)16-084).
135 136
2.2. Materials
137
(-)-Scopolamine hydrobromide, donepezil hydrochloride monohydrate, oleanolic acid,
138
rosmarinic acid and acetylcholinesterase (AChE) from Electrophorus electricus were
139
purchased from Sigma Chemical Co. (St. Louis, MO). The purities of the standards (oleanolic
140
acid and rosmarinic acid) for high performance liquid chromatography (HPLC) analysis were
141
all more than 98%. Anti-ERK, anti-phosphorylated ERK (p-ERK) and anti-CREB antibodies
142
were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). An anti-
143
phosphorylated CREB (p-CREB) antibody was purchased from Upstate Lake Placid (Lake
144
Placid, NY). All other materials were of the highest grades available and were obtained from
145
normal commercial sources. Donepezil, scopolamine and the ethanolic extract of D.
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moldavica were dissolved in a 0.9% physiological saline solution. 7
147 148
2.3. Preparation of the ethanolic extract of D. moldavica
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Dried leaves of D. moldavica were obtained from Professor Xiang-Qian Liu (School
150
of Pharmacy, Hunan University of Chinese Medicine, China). The material was authenticated
151
by Emeritus Professor Chang Soo Yook (Department of Oriental Pharmaceutical Science,
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College of Pharmacy, Kyung Hee University), and voucher specimen was deposited at the
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herbarium of the College of Pharmacy, Kyung Hee University (Voucher specimen No.:
154
KHUOPS-2017-31). To obtain EEDM, dried D. moldavica samples were extracted with 70%
155
ethanol twice for two hours in an ultrasonic bath. The obtained extract was then filtered,
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concentrated in a water bath under vacuum pressure, frozen, lyophilized (model FD-5N;
157
Eyela, Tokyo), and then stored at -20 οC until use.
158 159
2.4. HPLC analysis
160
2.4.1. Sample preparation and chromatographic conditions
161
An aliquot of 30.0 mg extract was accurately weighed and dissolved in 1 mL of
162
solution (H2O:acetonitrile:dimethylsulfoxide [DMSO] = 3:1:1) and diluted to 10 mg/mL. The
163
solution was then filtered through a 0.22 µm syringe filter before injection. HPLC analysis of
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EEDM was performed with a Dionex UltiMate 3000 UHPLC system (ThermoFisher,
165
Waltham, MA) equipped with a quaternary gradient pump (LPG-3400SD), an auto sampler
166
(ACC-3000), a column oven, and a diode array detector (DAD-3000). The sample were
167
separated on a YMC-Triart C18 column (250 mm × 4.5 mm, 5 µm) in gradient elution mode
168
with a mobile phase comprising 0.1% acetic acid in H2O (A) and acetonitrile (B) at a flow
169
rate of 1.0 mL/min. The column temperature was set to 35 ºC, and the sample injection
170
volume was 20 µL. The gradient program for rosmarinic acid of EEDM was as follows: 0–3
171
min, 100% (A); 3–8 min, 100-70% (A); 8–10 min, 70% (A); 10–25 min, 70–55% (A); 25–35 8
172
min, 55% (A). The gradient program for oleanolic acid of EEDM was as follows: 0–3 min,
173
100% (A); 3–10 min, 100-30% (A); 10–18 min, 30% (A); 18–22 min, 30–0% (A); 22–35 min,
174
0% (A). Analytes of rosmarinic acid and oleanolic acid of EEMD were detected at
175
wavelengths of 280 nm and 210 nm, respectively. The data were processed with Thermo
176
Scientific Chromeleon Chromatography Data System (CDS) software.
177 178
2.4.2. Quantification of rosmarinic acid and oleanolic acid
179
The reference standards, rosmarinic acid and oleanolic acid, were accurately weighed
180
and dissolved in H2O and DMSO, respectively. Each stock solution was transferred to an
181
Eppendorf tube and then diluted with H2O to obtain working solutions (600, 300, 100, 50, 10
182
µg/mL). A linearity test was established by analyzing a series of appropriate concentrations
183
prepared by diluting each working solution. A chromatogram was obtained for each
184
calibration curve by injecting the working solution into the column and performing HPLC
185
analysis. These peak data were plotted to draw calibration curves for quantitative analysis.
186
The rosmarinic acid and oleanolic acid contents were calculated by the calibration curve
187
equation as follows: rosmarinic acid, y = 0.3708x – 0.9849, R2 = 1; and oleanolic acid, y =
188
0.1825x + 0.8737, R2 = 0.9998. The average levels of rosmarinic acid and oleanolic acid in
189
the EEDM were approximately 31.24 ± 0.03 mg/g and 38.69 ± 0.26 mg/g, respectively (Fig.
190
1).
191 192
2.4. Behavioral tasks
193
2.4.1. Step-through passive avoidance task
194
The acquisition and retention assessments of the passive avoidance task were carried
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out using identical illuminated and non-illuminated compartments (20 cm × 20 cm × 20 cm)
196
containing a 50 W bulb, as described previously (Yi et al., 2018). The floor of the non9
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illuminated compartment was composed of 2 mm stainless-steel rods spaced 1 cm apart, and
198
the two compartments were separated by a guillotine door (5 cm × 5 cm).
199
The animals underwent two separate trials (an acquisition trial and a retention trial)
200
separated by 24 h. One hour before the acquisition trial, mice orally received either EEDM
201
(12.5, 25, 50, or 100 mg/kg) or donepezil (5 mg/kg). The control group received a 0.9%
202
saline vehicle solution rather than EEDM or donepezil. Thirty minutes after EEDM,
203
donepezil, or saline administration, the mice were treated with scopolamine (1 mg/kg,
204
intraperitoneally [i.p.]). For the acquisition trial, each mouse was initially placed in the
205
illuminated compartment, and 10 s later, the door between the two compartments was opened.
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When the mouse entered the non-illuminated compartment, the door was closed, and an
207
electrical foot shock (0.5 mA, 3 s) was delivered through the grid floor. The following scores
208
were awarded based on the response to electric shock: 3, jumping; 2, vocalization; 1,
209
flinching; 0, no response. The retention trial was conducted 24 h after the acquisition trial by
210
returning the mouse back to the illuminated compartment. The time required for the mouse to
211
enter the non-illuminated compartment after the door opened was defined as the latency in
212
both trials. The latencies were recorded for up to 300 s.
213
To investigate the effect of EEDM on learning and memory in unimpaired control
214
animals, EEDM was administered one hour before the acquisition trial. To avoid a ceiling
215
effect in the unimpaired animals, the intensity of the electrical foot shock was set at 0.25 mA
216
for 3 s. This lower intensity shock allowed for the examination of any potential enhancing
217
effects of EEDM.
218 219
2.4.2. Morris water maze task
220
The Morris water maze apparatus was a circular pool (90 cm in diameter and 45 cm in
221
height) with a featureless inner surface. The pool was filled to a depth of 30 cm with water 10
222
containing a black pigment. The tank was placed in a dimly lit, soundproof test room with
223
various visual cues. The pool was conceptually divided into quadrants. A black platform (6
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cm in diameter and 29 cm high) was then placed in one of the pool quadrants and submerged
225
1 cm below the water surface so that it was not visible. The test was conducted as described
226
previously (Park et al., 2012) with slight modifications.
227
The first experimental day was dedicated to swim training for 60 s in the absence of
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the platform. During the four subsequent days, the mice were given four training trials per
229
session per day in the presence of the platform. When a mouse located the platform, it was
230
permitted to remain on it for 10 s. If the mouse did not locate the platform within 60 s, it was
231
gently placed on the platform for 10 s. The animals were returned to their home cages and
232
allowed to dry under an infrared lamp after each trial. The time between the training trials
233
was 30 s. During each training trial session, the time taken to find the hidden platform
234
(latency) was recorded using a video camera-based EthoVision System XT (Noldus
235
Information Technology, Wageningen, Netherlands). For each training trial, the mice were
236
placed in the water in a randomly selected pool quadrant facing the pool wall. One day after
237
the last training trial session, the mice were underwent a probe trial session in which the
238
platform was removed from the pool, and the mice were allowed to search for it for 60 s. A
239
record was kept of the swimming time in the pool quadrant where the platform had been
240
located previously. EEDM (50 mg/kg, p.o.) or donepezil (5 mg/kg, p.o.) were administered
241
daily one hour before the first training trial of each session. Memory impairment was induced
242
by scopolamine (1 mg/kg, i.p.) 30 min after EEDM treatment. The control group only
243
received 0.9% saline (p.o.).
244 245 246
2.5. Western blot analysis After the administration of donepezil or EEDM with scopolamine, the mice were 11
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sacrificed via decapitation, and the brains were immediately removed. Isolated hippocampal
248
tissue was homogenized in ice-chilled Tris-HCl buffer solution (20 mM, pH 7.4) containing
249
protease and phosphatase inhibitors. The tissue lysate was centrifuged at 12,000 rpm at 4 ºC
250
for 20 min. The supernatant was quantified using the Bradford method using a Pierce BCA
251
protein assay kit (Thermo Scientific, PA), and 15 µg of protein was subjected to SDS-PAGE
252
(8% gel) under reducing conditions. Western blot analysis was conducted as described by a
253
previous study (Park et al., 2012). The proteins were transferred onto PVDF membranes in
254
transfer buffer and further separated at 100 V for 2 h at 4 °C to determine the p-ERK, CREB
255
and p-CREB levels. The membranes were incubated for 2 h with blocking solution (5% skim
256
milk) at 4 °C, followed by overnight incubation with a primary antibody (ERK, 1:3000; p-
257
ERK, 1:1000; CREB, 1:3000; and p-CREB, 1:1000). The membranes were then washed
258
twice with Tween 20/Tris-buffered saline (TTBS), incubated with a horseradish peroxidase-
259
conjugated secondary antibody for 2 h at room temperature, washed three times with TTBS,
260
and developed using enhanced chemiluminescence (Amersham Life Science, Arlington
261
Heights, IL). The immunoblots were imaged using a LAS-4000 mini imager (Fujifilm Life
262
Science USA, Stamford, CT) and analyzed using Multi Gauge version 3.2 (Fujifilm Holdings
263
Corporation, Tokyo, Japan). The phosphorylation level was determined by calculating the
264
ratio of phosphorylated protein to total protein on the same membrane.
265 266
2.6. AChE inhibition assay
267
Analysis of AChE activity was performed using acetylthiocholine iodide as a
268
synthetic substrate in a colorimetric assay, as described previously (Ellman et al., 1961).
269
AChE from E. electricus (electric eel) was used as the enzyme source for the assay. Each
270
drug was initially dissolved in DMSO and diluted to several concentrations immediately
271
before use. An aliquot of each diluted drug solution was then mixed with 640 µL of 12
272
phosphate buffer (0.1 M, pH 8.0), 25 µL of buffered Ellman’s reagent (10 mM 5,5-
273
dithiobis[2-nitrobenzoic acid], 15 mM sodium bicarbonate) and the enzyme source (100 µL);
274
the mixture was then preincubated at room temperature for 10 min. Ten minutes after the
275
addition of 5 µL of an acetylthiocholine iodide solution (75 mM), the absorbance was
276
measured at 410 nm using a UV spectrophotometer (OPTIZEN 2120UV, Mecasys Co., Ltd.,
277
Korea). The concentration of drug required to inhibit AChE activity by 50% (IC50) was
278
calculated using an enzyme inhibition dose-response curve. To exclude interference due to
279
the pigment of EEDM or donepezil, the same volume of solution containing the drug,
280
Ellman’s reagent, and the enzyme source without the acetylthiocholine iodide solution was
281
used as a blank.
282 283
2.7. Statistical analyses
284
All data analyses were done using GraphPad Prism Version 5.02 (GraphPad, La Jolla,
285
CA, USA). The results of the behavioral studies and Western blot analysis are expressed as
286
the mean ± standard error of the mean (SEM). The passive avoidance task latencies, Morris
287
water maze test probe trial swimming times, and Western blot immunoreactivity were
288
analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for
289
multiple comparisons. The Morris water maze test training trial latencies were analyzed using
290
two-way ANOVA followed by post hoc pairwise comparisons with a Bonferroni correction
291
for multiple comparisons (independent variable included day and treatment). Statistical
292
significance was set at p < 0.05.
293 294
3. Results
295 296
3.1. Effects of EEDM on scopolamine-induced memory impairment in the step-through 13
297
passive avoidance task
298
To investigate the effects of EEDM on the control mice (Fig. 2A and B), as well as the
299
scopolamine-induced amnesic mice (Fig. 2C and 2D), the step-through passive avoidance
300
task was conducted after a single administration of EEDM. In the case of the control mice
301
(which were not treated with scopolamine), there were no significant differences in the
302
latencies either in the acquisition or retention trials (one-way ANOVA, acquisition trial, F4, 44
303
= 0.537, P = 0.709; retention trial, F4, 44 = 1.624, P = 0.186; Fig. 2A), suggesting that the
304
single administration of EEDM or donepezil may not affect normal cognitive function. In the
305
case of the scopolamine-induced amnesic mice, significant group effects were observed in the
306
step-through latency in the retention trial (one-way ANOVA, F6, 59 = 12.81, P < 0.001, Fig.
307
2C). The mean step-through latency of the scopolamine (1 mg/kg, i.p.)-treated mice was
308
significantly lower than that of the control mice (P < 0.05). The reduction in latency was
309
significantly reversed by the administration of EEDM (25, 50 and 100 mg/kg, p.o.) in a dose-
310
dependent manner (P < 0.05), and donepezil, an AChE inhibitor, was used as a positive
311
control (P < 0.01). However, there were no significant intergroup differences in the step-
312
through latency during the acquisition trial (one-way ANOVA, F6, 59 = 1.549, P = 0.179, Fig.
313
2C). In addition, regarding the electric foot shock score, no significant differences were
314
observed in control mice (one-way ANOVA, F4,
315
scopolamine-induced amnesic mice (one-way ANOVA, F6, 59 = 0.756, P = 0.607, Fig. 2D),
316
indicating that the memory performance of each group may not be related to the sensitivity of
317
the mice to an electric foot shock.
44
= 0.234, P = 0.917, Fig. 2B) and
318 319
3.2. Effects of EEDM on scopolamine-induced memory impairment in the Morris water maze
320
task
321
The effect of EEDM on spatial learning and memory was evaluated using the Morris 14
322
water maze task. As shown in Fig. 3A, the scopolamine only-treated group (1 mg/kg, i.p.)
323
exhibited longer latencies than those exhibited by the vehicle-treated control group during the
324
training trials. However, the mean latencies of the scopolamine + EEDM (50 mg/kg, p.o.)-
325
treated and scopolamine + donepezil (5 mg/kg, p.o.)-treated groups were significantly shorter
326
than those of the scopolamine only-treated group on day 4 (two-way ANOVA, day, F3, 144 =
327
17.4, P < 0.001; treatment, F3, 144 = 17.4, P < 0.001). In the probe trial session, significant
328
intergroup differences were observed in the swimming times within the target quadrant that
329
previously contained the platform (one-way ANOVA, F3, 35 = 12.79, P = 0.002, Fig. 3B). The
330
reduced time spent within the target quadrant by scopolamine-treated mice was significantly
331
reversed by EEDM or donepezil (P < 0.05). In addition, there were no significant differences
332
observed in the swimming velocity across all groups (one-way ANOVA, F3, 33 = 2.126, P =
333
0.038, Fig. 3C).
334 335
3.3. Effect of EEDM on AChE activity
336
Previous studies have reported that compounds or extracts with AChE inhibitory
337
activity exhibit significant cognitive improving effects (Mathew and Subramanian, 2014).
338
Therefore, we investigated whether EEDM has inhibitory activity against AChE in vitro.
339
Donepezil is a well-known AChE inhibitor and showed dose-dependent inhibitory activity
340
against AChE. However, EEDM did not show any AChE inhibitory activity (Fig. 4).
341 342
3.4. EEDM activates ERK-CREB signaling cascade in the hippocampus
343
We next investigated whether EEDM activates memory-related signaling cascade
344
pathways in the hippocampal and cortical tissue of scopolamine-induced amnesic mice. As
345
shown in Fig. 5, compared to scopolamine treatment, the single oral administration of EEDM
346
(50 mg/kg) significantly increased the expression ratio of p-ERK/ERK (one-way ANOVA, F4, 15
347
15
348
0.008, Fig. 5B) in the hippocampus. Additionally, there was no effect of EEDM on
349
phosphorylated ERK or CREB expression in the cerebral cortex (data not shown). These
350
findings suggest that EEDM activates the ERK-CREB signaling cascade in the hippocampus,
351
which may lead to cognitive improvement.
= 6.265, P = 0.003, Fig. 5A) and p-CREB/CREB (one-way ANOVA, F4, 15 = 5.044, P =
352 353
4. Discussion
354
In the present study, we first found that EEDM ameliorated scopolamine-induced
355
memory decline in the step-through passive avoidance and Morris water maze tasks.
356
Interestingly, EEDM improved scopolamine-induced cognitive impairment but did not affect
357
the cognitive activity in control mice in the step-through passive avoidance task. It should be
358
noted that EEDM did not cause any changes in motor function, as measured by the swimming
359
speed in the Morris water maze task and the step-through latency in the acquisition trial of the
360
passive avoidance task. These results indicate that the memory-ameliorating effect of EEDM
361
on scopolamine-induced cognitive impairment was not related to changes in motor function,
362
sedation, or sensitivity to electricity. Martínez-Vázquez and colleagues reported that a single
363
intraperitoneal treatment with an aqueous extract of D. moldavica causes sedative effects
364
such as prolonged sleeping time, sedation, reduced locomotor activity, and motor
365
coordination impairment in mice (Martinez-Vazquez et al., 2012). We cannot rule out that the
366
differences between the results of our study and the aforementioned study are due to
367
differences in the method of administration or extract preparation. Furthermore, a previous
368
study found that a total flavonoid extract of D. moldavica prevents learning and memory
369
deficits without causing motor impairments in APP/PS1 transgenic mice (Liu et al., 2018),
370
which supports our results. Collectively, these data indicate that D. moldavica may be a
371
potential agent for ameliorating cognitive dysfunction. 16
372
Previous phytochemical studies have revealed that oleanolic acid, rosmarinic acid,
373
chlorogenic acid, and apigenin are the major flavonoid compounds of D. moldavica
374
(Dastmalchi et al., 2007; Li et al., 2016). We also observed that EEDM contains rosmarinic
375
acid (31.24 ± 0.03 mg/g extract) and oleanolic acid (38.69 ± 0.26 mg/g extract). Mounting
376
evidence suggests that oleanolic acid ameliorates β-amyloid or scopolamine-induced
377
cognitive impairment through the activation of the TrkB-BDNF signaling cascade (Jeon et al.,
378
2017; Wang et al., 2018). Further, rosmarinic acid exhibits protective effects against Aβ-
379
induced cognitive impairment in the CNS (Alkam et al., 2007). Hasanein and Mahtaj (2015)
380
also reported that rosmarinic acid has an ameliorative effect on scopolamine-induced learning
381
and memory impairment in rats model (Hasanein and Mahtaj, 2015). Chlorogenic acid has
382
been reported to have neuroprotective and anti-amnesic effects against scopolamine-induced
383
amnesia in mice (Kwon et al., 2010). Another study indicated that apigenin improves
384
cognitive dysfunction and neuroinflammation via the upregulation of the ERK/CREB
385
pathway in APP/PS1 transgenic AD mice (Zhao et al., 2013). Together, these results suggest
386
that the memory-ameliorating effect of EEDM against scopolamine-induced impairment may
387
be attributed to the presence of these compounds.
388
It is well known that the cholinergic neurotransmission system in the basal forebrain
389
plays a critical role in learning and memory. Cholinergic transmission is mainly inactivated
390
by acetylcholine hydrolysis through AChE enzyme activity, which is responsible for the
391
degradation of acetylcholine into acetate and choline in the synaptic cleft (Ballard et al.,
392
2005). Excessive AChE activity leads to a persistent acetylcholine shortage and cognitive
393
dysfunction (Pepeu and Giovannini, 2010). Therefore, many researchers have focused on
394
searching for substances that can improve cognitive performance through the inhibition of
395
AChE activity. We also tested if EEDM could serve this purpose, as a previous reported study
396
indicated that Dracocephalum multicaule inhibits AChE enzyme activity (Mandegary et al., 17
397
2014). However, our results showed that EEDM does not have inhibitory activity against
398
AChE in an in vitro assay. These results suggest that the memory-ameliorating effect of
399
EEDM is not related to the inhibition of the AChE enzyme.
400
CREB is one of the key signaling molecules involved in learning and memory.
401
CREB is a transcription factor that functions as a molecular switch to control synaptic
402
plasticity and memory formation (Alberini, 2009). Meanwhile, the activation of CREB is
403
mediated by phosphorylation at serine 133, which can be controlled by ERK, Akt (also
404
known as protein kinase B), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and
405
protein kinase A (PKA) (Deak et al., 1998; Du and Montminy, 1998; Sun et al., 1994). ERK
406
activation is also highly associated with the development of several forms of memory,
407
including recognition and spatial memory (Kim et al., 2012). Previous studies have
408
confirmed the relationship between the ERK-CREB signaling pathway and memory
409
processing, suggesting that the activation of the ERK-CREB pathway possibly promotes
410
memory function (Davis et al., 2000; Peng et al., 2010). Our results showed that EEDM
411
significantly increased the phosphorylation levels of ERK and CREB in the hippocampus, but
412
not in the cerebral cortex. Notably, EEDM did not show any significant effects on the
413
phosphorylation levels of Akt, CaMKII or PKA in the hippocampus or cerebral cortex (data
414
not shown). These data indicate that the memory-ameliorating effect of EEDM may be due to
415
the activation of the ERK-CREB signaling cascade in the hippocampus. A previous study also
416
found that the flavonoid extract of D. moldavica effectively protects neurons against Aβ
417
accumulation and memory impairment by ERK-CREB pathway activation (Liu et al., 2018).
418
In addition, numerous studies have reported that activation of the ERK-CREB pathway is
419
associated with the enhancement of cognitive function as a result of the major constituent of
420
EEDM, including apigenin and oleanolic acid (Yi et al., 2014; Zhao et al., 2013). Therefore,
421
we suggest that the ERK-CREB signaling pathway is key mediator of the memory18
422
ameliorating effects observed in this study.
423
In conclusion, we demonstrated that EEDM effectively ameliorates scopolamine-
424
induced memory impairment in mice as measured by the passive avoidance and Morris water
425
maze tasks. Furthermore, EEDM may attenuate memory impairment through the activation of
426
the ERK-CREB pathway. These results suggest that D. moldavica may be used as a potential
427
therapeutic
428
neurodegenerative diseases.
agent
for
treating
cognitive
impairment
associated
with
various
429 430
Acknowledgments
431
This study was supported by the National Research Foundation of Korea (NRF) grant
432
funded by the Ministry of Science and ICT (NRF-2017R1C1B5017445; NRF-
433
2017R1A5A2014768).
434 435
Author's contributions
436
The study was conceived and designed by J.H.R. and S.J.P. Behavioral studies were
437
conducted by P.D., H.J.B. and H.P. Immunoblotting assays were performed by H.J.B, S.K.
438
and J.W.C. EEDM sample was prepared and standardized by X.L., S.K. and D.H.K.. The
439
manuscript was written by P.D., J.H.R. and S.J.P.
440 441
Conflict of interests
442
The authors declare that there is no conflict of interest.
443 444
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disease
like
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impairments
557
24
and
aberrant
synaptic
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558 559
Figure legends
560 561
Figure 1. HPLC analysis of EEDM and standard compounds with detector responses at 280
562
and 210 nm. Detector responses were as follows: (A) Rosmaric acid at 280 nm, (B) EEDM at
563
280 nm, (C) oleanolic acid at 210 nm, and (D) EEDM at 210 nm. EEDM, ethanolic extract of
564
D. moldavica; HPLC, high performance liquid chromatography.
565 566
Figure 2. Effects of EEDM on unimpaired control mice and mice with scopolamine-induced
567
memory impairment in the passive avoidance task. (A) Step-through latency and (B)
568
electrosensitivity in unimpaired control mice; (C) Step-through latency and (D)
569
electrosensitivity in scopolamine-induced amnesic mice. The data represent the means ±
570
SEM (n = 9 - 10 per group). *p < 0.05 versus the vehicle-treated controls, #p < 0.05 versus the
571
scopolamine-treated group. Con, control; DNZ, donepezil; EEDM, ethanolic extract of D.
572
moldavica.
573 574
Figure 3. Effects of EEDM on scopolamine-induced memory impairment in the Morris water
575
maze task. (A) Latencies during the training trial sessions, (B) swimming time spent in the
576
target quadrant during the probe trial session and (C) swimming velocity during the probe
577
trial session. The data represent the means ± SEM (n = 8 - 9 per group)
578
the vehicle-treated controls; #p < 0.05, ##p < 0.01 versus the scopolamine-treated group. DNZ,
579
donepezil; EEDM, ethanolic extract of D. moldavica.
***
p < 0.001 versus
580 581
Figure 4. Effects of EEDM and donepezil on acetylcholinesterase (AChE) activity in vitro.
582
AChE activity was measured using a colorimetric assay using acetylthiocholine iodide as a
583
synthetic substrate. The AChE activity of each sample was observed three times. EEDM, 25
584
ethanolic extract of D. moldavica.
585 586
Figure 5. Effects of EEDM on ERK and CREB signaling cascades in the hippocampus of
587
scopolamine-induced amnesic mice. The immunoreactivity of phosphorylated ERK (pERK)
588
and ERK (A) and phosphorylated CREB (pCREB) and CREB (B) in the hippocampus were
589
quantified. The data represent the mean ± SEM. (n = 3-4/group) #p < 0.05 versus the
590
scopolamine-treated group. DNZ, donepezil; EEDM, ethanolic extract of D. moldavica ; Sco,
591
scopolamine.
26
S0378-8741(19)31446-1
DOI:
https://doi.org/10.1016/j.jep.2020.112651
Reference:
JEP 112651
To appear in:
Journal of Ethnopharmacology
Received Date: 10 April 2019 Revised Date:
3 January 2020
Accepted Date: 2 February 2020
Please cite this article as: Deepa, P., Bae, H.J., Park, H.-B., Kim, S.-Y., Kim, S., Choi, J.W., Kim, D.H., Liu, X.-Q., Ryu, J.H., Park, S.J., Dracocephalum moldavica attenuates scopolamine-induced cognitive impairment through activation of hippocampal ERK-CREB signaling in mice, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/j.jep.2020.112651. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Graphical abstract
1
Dracocephalum moldavica attenuates scopolamine-induced cognitive impairment
2
through activation of hippocampal ERK-CREB signaling in mice
3 4
Ponnuvel Deepa1, Ho Jung Bae2, Hyeon-Bae Park1, So-Yeon Kim1, Songmun Kim1, Ji
5
Woong Choi5, Dong Hyun Kim6, Xiang-Qian Liu4, Jong Hoon Ryu2, 3, *, Se Jin Park1, *
6 7
1
8
Chuncheon, Republic of Korea
9
2
School of Natural Resources and Environmental Sciences, Kangwon National University,
Department of Life and Nanopharmaceutical Sciences and
3
Department of Oriental
10
Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, Republic of
11
Korea
12
4
13
5
14
Pharmaceutical Sciences, Gachon University, Incheon, Republic of Korea
15
6
16
Convergence Bio-Health, Dong-A University, Busan, Republic of Korea
School of Pharmacy, Hunan University of Chinese Medicine, Changsha, China Laboratory of Neuropharmacology, College of Pharmacy and Gachon Institute of
Department of Medicinal Biotechnology, College of Health Sciences and Institute of
17 18
*
19
Se Jin Park at School of Natural Resources and Environmental Sciences, Kangwon National
20
University, Chuncheon, Republic of Korea; [email protected]
21
J.H. Ryu at Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung
22
Hee University, Seoul, Republic of Korea; [email protected]
Corresponding Authors
23 24
1
25
Authors E-mail:
26
[email protected] (P. Deepa); [email protected] (H.J. Bae)
27
[email protected] (Hyun-Bae Park); [email protected] (So-Yeon Kim)
28
[email protected] (Songmun Kim); [email protected] (Xiang-Qian Liu)
29
[email protected] (Dong Hyun Kim); [email protected] (Ji Woong Choi)
30
[email protected] (Jong Hoon Ryu); [email protected] (Se Jin Park)
31 32
Running title: Memory-ameliorating effect of Dracocephalum moldavica
2
33
List of abbreviations
34
AChE, acetylcholinesterase
35
Aβ, amyloid beta
36
AD, Alzheimer’s disease
37
BDNF, brain-derived neurotrophic factor
38
CaMKII, Ca2+/calmodulin-dependent protein kinase II
39
CNS, central nervous system
40
CREB, cAMP response element-binding protein
41
DNZ, donepezil
42
EEDM, ethanolic extract of Dracocephalum moldavica
43
ERK, extracellular signal regulated kinase
44
PKA, protein kinase A
45
3
46
Abstract
47
Ethnopharmacological relevance: Dracocephalum moldavica (Moldavian balm) has been
48
traditionally used for the treatment of intellectual disabilities, migraines and cardiovascular
49
problems in East Asia. Recent scientific studies have demonstrated the usefulness of this
50
plant to treat neurodegenerative disorders, including Alzheimer’s disease.
51
Aim of the study: This study aimed to investigate the effects of the ethanolic extract of D.
52
moldavica leaves (EEDM) on scopolamine-induced cognitive impairment in mice and the
53
underlying mechanisms of action.
54
Materials and methods: The behavioral effects of EEDM were examined using the step-through
55
passive avoidance and Morris water maze tasks. To elucidate the underlying mechanism, we
56
tested whether EEDM affects acetylcholinesterase activity and the expression of memory-
57
related signaling molecules including extracellular signal-regulated kinase (ERK) and cAMP
58
response element-binding protein (CREB) in the hippocampus.
59
Results: EEDM (25, 50 or 100 mg/kg) significantly ameliorated the scopolamine-induced
60
step-through latency reduction in the passive avoidance task in mice. In the Morris water
61
maze task, EEDM (50 mg/kg) significantly attenuated scopolamine-induced memory
62
impairment. Furthermore, the administration of EEDM increased the phosphorylation levels
63
of ERK and CREB in the hippocampus but did not alter acetylcholinesterase activity.
64
Conclusions: These findings suggest that EEDM significantly attenuates scopolamine-
65
induced memory impairment in mice and may be a promising therapeutic agent for
66
improving memory impairment.
67 68
Keywords: Dracocephalum moldavica; memory impairment; Alzheimer’s disease;
69
scopolamine; extracellular signal regulated kinase; cAMP response element-binding protein
70 71
4
72
1. Introduction
73
Alzheimer’s disease (AD) is mainly characterized by memory deficits and mental
74
dysfunction; the former is known to be mainly correlated with declines in cholinergic
75
neurotransmission systems (Francis et al., 1999). Behavioral studies have shown that anti-
76
cholinergic drugs impair cognitive function in healthy humans and animals (Atri et al., 2004;
77
Flood and Cherkin, 1986). Accordingly, blockade of the cholinergic system with muscarinic
78
cholinergic receptor antagonists (e.g. scopolamine) is widely used to induce cognitive
79
impairment (Klinkenberg and Blokland, 2010). Moreover, several synthetic drugs, including
80
cholinesterase inhibitors, have been used for cognitive enhancement. Although synthetic
81
memory-enhancing drugs effectively improve memory performance, they have several
82
adverse effects, such as nausea, vomiting, diarrhea and anorexia (Gauthier, 2001). Thus,
83
many studies have focused on the identification of novel drugs, particularly herbal plants, to
84
treat various neurodegenerative diseases, including AD.
85
Dracocephalum moldavica L. (Lamiaceae, Labiatae) is a perennial aromatic herb
86
native to central Asia, northern China, and eastern and central Europe, and is commonly
87
referred to as Moldavian balm. Because it is naturally warm and fragrant, D. moldavica can
88
affect the central nervous system (CNS), cardiac tissues, and blood circulation (Liu et al.,
89
2018). Accordingly, D. moldavica has been traditionally used for the treatment of heart
90
disease, blood pressure, angina, atherosclerosis, neuralgia, migraines, headaches and
91
toothaches (Dastmalchi et al., 2007; Liu et al., 2018; Maimaitiyiming et al., 2014; Zhao et al.,
92
2017). Additionally, recent studies have also confirmed that D. moldavica has various
93
pharmacological effects on the CNS, such as neuroprotection against rat cerebral ischemia
94
reperfusion injury (Jia et al., 2017; Zeng et al., 2018), anti-oxidative and anti-inflammatory
95
properties in an animal model of AD (Liu et al., 2018) and the promotion of prolonged
96
pentobarbital-induced sleeping time and sedation in mice (Martinez-Vazquez et al., 2012). 5
97
Furthermore, phytochemical studies have revealed that D. moldavica primarily contains
98
rosmarinic acid, oleanolic acid, chlorogenic acid, ferulic acid, caffeic acid, p-coumaric acid,
99
apigenin, quercetin, acacetin, tilianin and luteolin (Li et al., 2016). We and several groups
100
have reported that scopolamine-induced cognitive impairment is ameliorated by oleanolic
101
acid (Jeon et al., 2017), rosmarinic acid (Qu et al., 2017) and chlorogenic acid (Kwon et al.,
102
2010) which are documented constituents of D. moldavica.
103
Extracellular signal-regulated kinase (ERK) and cAMP response element-binding
104
protein (CREB) signaling molecules are known to be involved in cognitive functions. ERK
105
belongs to the mitogen-activated protein kinase family member and activates CREB, which
106
regulates cellular processes for the regulation of long-term synaptic plasticity and the
107
stabilization of new memories (Adams and Sweatt, 2002; Kelleher et al., 2004). Multiple
108
studies have confirmed that improvements in cognitive abilities are facilitated by the
109
activation of ERK signaling (Ciccarelli and Giustetto, 2014; Kim et al., 2012). CREB is a
110
transcription factor that binds to the promoter regions of many neuronal genes associated
111
with learning, memory and synaptic plasticity (Alberini, 2009). Thus, the activation of the
112
ERK-CREB signaling cascade is necessary for the formation and storage of memories in the
113
hippocampus. It should be noted that a total flavonoid extract of D. moldavica has been
114
reported to attenuate β-amyloid-induced neurotoxicity through the activation of neurotrophic
115
pathways, including the ERK-CREB-brain-derived neurotrophic factor (BDNF) pathway (Liu
116
et al., 2018).
117
Based on previous studies, we hypothesized that D. moldavica may cure cognitive
118
disorders by targeting ERK/CREB signaling. However, no reports have described the
119
memory-ameliorating effect of D. moldavica on cognitive impairments due to cholinergic
120
blockade. Hence, the aim of this study was to investigate whether the ethanolic extract of D.
121
moldavica (EEDM) attenuates the scopolamine-induced cognitive impairment in mice using 6
122
the passive avoidance and Morris water maze tasks. We also investigated whether EEDM
123
affects the phosphorylation levels of ERK and CREB in the hippocampus.
124 125
2. Materials and methods
126
2.1. Animals
127
Male CD1 ICR mice (6 weeks old, 25–30 g) were purchased from the Orient Co. Ltd.,
128
a branch of Charles River Laboratories (Seoul, Korea). The mice were housed in groups of 5
129
per cage, provided with ad libitum access to food and water, and kept under a 12 h light/dark
130
cycle (lights on 07:00–19:00) at a constant temperature (23 ± 1 ºC) and relative humidity (60
131
± 10%). Animal treatment and maintenance were carried out in accordance with the
132
Principles of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and with
133
the Animal Care and Use Guidelines issued by Kyung Hee University, Republic of Korea
134
(approval number: KHUASP(SE)16-084).
135 136
2.2. Materials
137
(-)-Scopolamine hydrobromide, donepezil hydrochloride monohydrate, oleanolic acid,
138
rosmarinic acid and acetylcholinesterase (AChE) from Electrophorus electricus were
139
purchased from Sigma Chemical Co. (St. Louis, MO). The purities of the standards (oleanolic
140
acid and rosmarinic acid) for high performance liquid chromatography (HPLC) analysis were
141
all more than 98%. Anti-ERK, anti-phosphorylated ERK (p-ERK) and anti-CREB antibodies
142
were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). An anti-
143
phosphorylated CREB (p-CREB) antibody was purchased from Upstate Lake Placid (Lake
144
Placid, NY). All other materials were of the highest grades available and were obtained from
145
normal commercial sources. Donepezil, scopolamine and the ethanolic extract of D.
146
moldavica were dissolved in a 0.9% physiological saline solution. 7
147 148
2.3. Preparation of the ethanolic extract of D. moldavica
149
Dried leaves of D. moldavica were obtained from Professor Xiang-Qian Liu (School
150
of Pharmacy, Hunan University of Chinese Medicine, China). The material was authenticated
151
by Emeritus Professor Chang Soo Yook (Department of Oriental Pharmaceutical Science,
152
College of Pharmacy, Kyung Hee University), and voucher specimen was deposited at the
153
herbarium of the College of Pharmacy, Kyung Hee University (Voucher specimen No.:
154
KHUOPS-2017-31). To obtain EEDM, dried D. moldavica samples were extracted with 70%
155
ethanol twice for two hours in an ultrasonic bath. The obtained extract was then filtered,
156
concentrated in a water bath under vacuum pressure, frozen, lyophilized (model FD-5N;
157
Eyela, Tokyo), and then stored at -20 οC until use.
158 159
2.4. HPLC analysis
160
2.4.1. Sample preparation and chromatographic conditions
161
An aliquot of 30.0 mg extract was accurately weighed and dissolved in 1 mL of
162
solution (H2O:acetonitrile:dimethylsulfoxide [DMSO] = 3:1:1) and diluted to 10 mg/mL. The
163
solution was then filtered through a 0.22 µm syringe filter before injection. HPLC analysis of
164
EEDM was performed with a Dionex UltiMate 3000 UHPLC system (ThermoFisher,
165
Waltham, MA) equipped with a quaternary gradient pump (LPG-3400SD), an auto sampler
166
(ACC-3000), a column oven, and a diode array detector (DAD-3000). The sample were
167
separated on a YMC-Triart C18 column (250 mm × 4.5 mm, 5 µm) in gradient elution mode
168
with a mobile phase comprising 0.1% acetic acid in H2O (A) and acetonitrile (B) at a flow
169
rate of 1.0 mL/min. The column temperature was set to 35 ºC, and the sample injection
170
volume was 20 µL. The gradient program for rosmarinic acid of EEDM was as follows: 0–3
171
min, 100% (A); 3–8 min, 100-70% (A); 8–10 min, 70% (A); 10–25 min, 70–55% (A); 25–35 8
172
min, 55% (A). The gradient program for oleanolic acid of EEDM was as follows: 0–3 min,
173
100% (A); 3–10 min, 100-30% (A); 10–18 min, 30% (A); 18–22 min, 30–0% (A); 22–35 min,
174
0% (A). Analytes of rosmarinic acid and oleanolic acid of EEMD were detected at
175
wavelengths of 280 nm and 210 nm, respectively. The data were processed with Thermo
176
Scientific Chromeleon Chromatography Data System (CDS) software.
177 178
2.4.2. Quantification of rosmarinic acid and oleanolic acid
179
The reference standards, rosmarinic acid and oleanolic acid, were accurately weighed
180
and dissolved in H2O and DMSO, respectively. Each stock solution was transferred to an
181
Eppendorf tube and then diluted with H2O to obtain working solutions (600, 300, 100, 50, 10
182
µg/mL). A linearity test was established by analyzing a series of appropriate concentrations
183
prepared by diluting each working solution. A chromatogram was obtained for each
184
calibration curve by injecting the working solution into the column and performing HPLC
185
analysis. These peak data were plotted to draw calibration curves for quantitative analysis.
186
The rosmarinic acid and oleanolic acid contents were calculated by the calibration curve
187
equation as follows: rosmarinic acid, y = 0.3708x – 0.9849, R2 = 1; and oleanolic acid, y =
188
0.1825x + 0.8737, R2 = 0.9998. The average levels of rosmarinic acid and oleanolic acid in
189
the EEDM were approximately 31.24 ± 0.03 mg/g and 38.69 ± 0.26 mg/g, respectively (Fig.
190
1).
191 192
2.4. Behavioral tasks
193
2.4.1. Step-through passive avoidance task
194
The acquisition and retention assessments of the passive avoidance task were carried
195
out using identical illuminated and non-illuminated compartments (20 cm × 20 cm × 20 cm)
196
containing a 50 W bulb, as described previously (Yi et al., 2018). The floor of the non9
197
illuminated compartment was composed of 2 mm stainless-steel rods spaced 1 cm apart, and
198
the two compartments were separated by a guillotine door (5 cm × 5 cm).
199
The animals underwent two separate trials (an acquisition trial and a retention trial)
200
separated by 24 h. One hour before the acquisition trial, mice orally received either EEDM
201
(12.5, 25, 50, or 100 mg/kg) or donepezil (5 mg/kg). The control group received a 0.9%
202
saline vehicle solution rather than EEDM or donepezil. Thirty minutes after EEDM,
203
donepezil, or saline administration, the mice were treated with scopolamine (1 mg/kg,
204
intraperitoneally [i.p.]). For the acquisition trial, each mouse was initially placed in the
205
illuminated compartment, and 10 s later, the door between the two compartments was opened.
206
When the mouse entered the non-illuminated compartment, the door was closed, and an
207
electrical foot shock (0.5 mA, 3 s) was delivered through the grid floor. The following scores
208
were awarded based on the response to electric shock: 3, jumping; 2, vocalization; 1,
209
flinching; 0, no response. The retention trial was conducted 24 h after the acquisition trial by
210
returning the mouse back to the illuminated compartment. The time required for the mouse to
211
enter the non-illuminated compartment after the door opened was defined as the latency in
212
both trials. The latencies were recorded for up to 300 s.
213
To investigate the effect of EEDM on learning and memory in unimpaired control
214
animals, EEDM was administered one hour before the acquisition trial. To avoid a ceiling
215
effect in the unimpaired animals, the intensity of the electrical foot shock was set at 0.25 mA
216
for 3 s. This lower intensity shock allowed for the examination of any potential enhancing
217
effects of EEDM.
218 219
2.4.2. Morris water maze task
220
The Morris water maze apparatus was a circular pool (90 cm in diameter and 45 cm in
221
height) with a featureless inner surface. The pool was filled to a depth of 30 cm with water 10
222
containing a black pigment. The tank was placed in a dimly lit, soundproof test room with
223
various visual cues. The pool was conceptually divided into quadrants. A black platform (6
224
cm in diameter and 29 cm high) was then placed in one of the pool quadrants and submerged
225
1 cm below the water surface so that it was not visible. The test was conducted as described
226
previously (Park et al., 2012) with slight modifications.
227
The first experimental day was dedicated to swim training for 60 s in the absence of
228
the platform. During the four subsequent days, the mice were given four training trials per
229
session per day in the presence of the platform. When a mouse located the platform, it was
230
permitted to remain on it for 10 s. If the mouse did not locate the platform within 60 s, it was
231
gently placed on the platform for 10 s. The animals were returned to their home cages and
232
allowed to dry under an infrared lamp after each trial. The time between the training trials
233
was 30 s. During each training trial session, the time taken to find the hidden platform
234
(latency) was recorded using a video camera-based EthoVision System XT (Noldus
235
Information Technology, Wageningen, Netherlands). For each training trial, the mice were
236
placed in the water in a randomly selected pool quadrant facing the pool wall. One day after
237
the last training trial session, the mice were underwent a probe trial session in which the
238
platform was removed from the pool, and the mice were allowed to search for it for 60 s. A
239
record was kept of the swimming time in the pool quadrant where the platform had been
240
located previously. EEDM (50 mg/kg, p.o.) or donepezil (5 mg/kg, p.o.) were administered
241
daily one hour before the first training trial of each session. Memory impairment was induced
242
by scopolamine (1 mg/kg, i.p.) 30 min after EEDM treatment. The control group only
243
received 0.9% saline (p.o.).
244 245 246
2.5. Western blot analysis After the administration of donepezil or EEDM with scopolamine, the mice were 11
247
sacrificed via decapitation, and the brains were immediately removed. Isolated hippocampal
248
tissue was homogenized in ice-chilled Tris-HCl buffer solution (20 mM, pH 7.4) containing
249
protease and phosphatase inhibitors. The tissue lysate was centrifuged at 12,000 rpm at 4 ºC
250
for 20 min. The supernatant was quantified using the Bradford method using a Pierce BCA
251
protein assay kit (Thermo Scientific, PA), and 15 µg of protein was subjected to SDS-PAGE
252
(8% gel) under reducing conditions. Western blot analysis was conducted as described by a
253
previous study (Park et al., 2012). The proteins were transferred onto PVDF membranes in
254
transfer buffer and further separated at 100 V for 2 h at 4 °C to determine the p-ERK, CREB
255
and p-CREB levels. The membranes were incubated for 2 h with blocking solution (5% skim
256
milk) at 4 °C, followed by overnight incubation with a primary antibody (ERK, 1:3000; p-
257
ERK, 1:1000; CREB, 1:3000; and p-CREB, 1:1000). The membranes were then washed
258
twice with Tween 20/Tris-buffered saline (TTBS), incubated with a horseradish peroxidase-
259
conjugated secondary antibody for 2 h at room temperature, washed three times with TTBS,
260
and developed using enhanced chemiluminescence (Amersham Life Science, Arlington
261
Heights, IL). The immunoblots were imaged using a LAS-4000 mini imager (Fujifilm Life
262
Science USA, Stamford, CT) and analyzed using Multi Gauge version 3.2 (Fujifilm Holdings
263
Corporation, Tokyo, Japan). The phosphorylation level was determined by calculating the
264
ratio of phosphorylated protein to total protein on the same membrane.
265 266
2.6. AChE inhibition assay
267
Analysis of AChE activity was performed using acetylthiocholine iodide as a
268
synthetic substrate in a colorimetric assay, as described previously (Ellman et al., 1961).
269
AChE from E. electricus (electric eel) was used as the enzyme source for the assay. Each
270
drug was initially dissolved in DMSO and diluted to several concentrations immediately
271
before use. An aliquot of each diluted drug solution was then mixed with 640 µL of 12
272
phosphate buffer (0.1 M, pH 8.0), 25 µL of buffered Ellman’s reagent (10 mM 5,5-
273
dithiobis[2-nitrobenzoic acid], 15 mM sodium bicarbonate) and the enzyme source (100 µL);
274
the mixture was then preincubated at room temperature for 10 min. Ten minutes after the
275
addition of 5 µL of an acetylthiocholine iodide solution (75 mM), the absorbance was
276
measured at 410 nm using a UV spectrophotometer (OPTIZEN 2120UV, Mecasys Co., Ltd.,
277
Korea). The concentration of drug required to inhibit AChE activity by 50% (IC50) was
278
calculated using an enzyme inhibition dose-response curve. To exclude interference due to
279
the pigment of EEDM or donepezil, the same volume of solution containing the drug,
280
Ellman’s reagent, and the enzyme source without the acetylthiocholine iodide solution was
281
used as a blank.
282 283
2.7. Statistical analyses
284
All data analyses were done using GraphPad Prism Version 5.02 (GraphPad, La Jolla,
285
CA, USA). The results of the behavioral studies and Western blot analysis are expressed as
286
the mean ± standard error of the mean (SEM). The passive avoidance task latencies, Morris
287
water maze test probe trial swimming times, and Western blot immunoreactivity were
288
analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for
289
multiple comparisons. The Morris water maze test training trial latencies were analyzed using
290
two-way ANOVA followed by post hoc pairwise comparisons with a Bonferroni correction
291
for multiple comparisons (independent variable included day and treatment). Statistical
292
significance was set at p < 0.05.
293 294
3. Results
295 296
3.1. Effects of EEDM on scopolamine-induced memory impairment in the step-through 13
297
passive avoidance task
298
To investigate the effects of EEDM on the control mice (Fig. 2A and B), as well as the
299
scopolamine-induced amnesic mice (Fig. 2C and 2D), the step-through passive avoidance
300
task was conducted after a single administration of EEDM. In the case of the control mice
301
(which were not treated with scopolamine), there were no significant differences in the
302
latencies either in the acquisition or retention trials (one-way ANOVA, acquisition trial, F4, 44
303
= 0.537, P = 0.709; retention trial, F4, 44 = 1.624, P = 0.186; Fig. 2A), suggesting that the
304
single administration of EEDM or donepezil may not affect normal cognitive function. In the
305
case of the scopolamine-induced amnesic mice, significant group effects were observed in the
306
step-through latency in the retention trial (one-way ANOVA, F6, 59 = 12.81, P < 0.001, Fig.
307
2C). The mean step-through latency of the scopolamine (1 mg/kg, i.p.)-treated mice was
308
significantly lower than that of the control mice (P < 0.05). The reduction in latency was
309
significantly reversed by the administration of EEDM (25, 50 and 100 mg/kg, p.o.) in a dose-
310
dependent manner (P < 0.05), and donepezil, an AChE inhibitor, was used as a positive
311
control (P < 0.01). However, there were no significant intergroup differences in the step-
312
through latency during the acquisition trial (one-way ANOVA, F6, 59 = 1.549, P = 0.179, Fig.
313
2C). In addition, regarding the electric foot shock score, no significant differences were
314
observed in control mice (one-way ANOVA, F4,
315
scopolamine-induced amnesic mice (one-way ANOVA, F6, 59 = 0.756, P = 0.607, Fig. 2D),
316
indicating that the memory performance of each group may not be related to the sensitivity of
317
the mice to an electric foot shock.
44
= 0.234, P = 0.917, Fig. 2B) and
318 319
3.2. Effects of EEDM on scopolamine-induced memory impairment in the Morris water maze
320
task
321
The effect of EEDM on spatial learning and memory was evaluated using the Morris 14
322
water maze task. As shown in Fig. 3A, the scopolamine only-treated group (1 mg/kg, i.p.)
323
exhibited longer latencies than those exhibited by the vehicle-treated control group during the
324
training trials. However, the mean latencies of the scopolamine + EEDM (50 mg/kg, p.o.)-
325
treated and scopolamine + donepezil (5 mg/kg, p.o.)-treated groups were significantly shorter
326
than those of the scopolamine only-treated group on day 4 (two-way ANOVA, day, F3, 144 =
327
17.4, P < 0.001; treatment, F3, 144 = 17.4, P < 0.001). In the probe trial session, significant
328
intergroup differences were observed in the swimming times within the target quadrant that
329
previously contained the platform (one-way ANOVA, F3, 35 = 12.79, P = 0.002, Fig. 3B). The
330
reduced time spent within the target quadrant by scopolamine-treated mice was significantly
331
reversed by EEDM or donepezil (P < 0.05). In addition, there were no significant differences
332
observed in the swimming velocity across all groups (one-way ANOVA, F3, 33 = 2.126, P =
333
0.038, Fig. 3C).
334 335
3.3. Effect of EEDM on AChE activity
336
Previous studies have reported that compounds or extracts with AChE inhibitory
337
activity exhibit significant cognitive improving effects (Mathew and Subramanian, 2014).
338
Therefore, we investigated whether EEDM has inhibitory activity against AChE in vitro.
339
Donepezil is a well-known AChE inhibitor and showed dose-dependent inhibitory activity
340
against AChE. However, EEDM did not show any AChE inhibitory activity (Fig. 4).
341 342
3.4. EEDM activates ERK-CREB signaling cascade in the hippocampus
343
We next investigated whether EEDM activates memory-related signaling cascade
344
pathways in the hippocampal and cortical tissue of scopolamine-induced amnesic mice. As
345
shown in Fig. 5, compared to scopolamine treatment, the single oral administration of EEDM
346
(50 mg/kg) significantly increased the expression ratio of p-ERK/ERK (one-way ANOVA, F4, 15
347
15
348
0.008, Fig. 5B) in the hippocampus. Additionally, there was no effect of EEDM on
349
phosphorylated ERK or CREB expression in the cerebral cortex (data not shown). These
350
findings suggest that EEDM activates the ERK-CREB signaling cascade in the hippocampus,
351
which may lead to cognitive improvement.
= 6.265, P = 0.003, Fig. 5A) and p-CREB/CREB (one-way ANOVA, F4, 15 = 5.044, P =
352 353
4. Discussion
354
In the present study, we first found that EEDM ameliorated scopolamine-induced
355
memory decline in the step-through passive avoidance and Morris water maze tasks.
356
Interestingly, EEDM improved scopolamine-induced cognitive impairment but did not affect
357
the cognitive activity in control mice in the step-through passive avoidance task. It should be
358
noted that EEDM did not cause any changes in motor function, as measured by the swimming
359
speed in the Morris water maze task and the step-through latency in the acquisition trial of the
360
passive avoidance task. These results indicate that the memory-ameliorating effect of EEDM
361
on scopolamine-induced cognitive impairment was not related to changes in motor function,
362
sedation, or sensitivity to electricity. Martínez-Vázquez and colleagues reported that a single
363
intraperitoneal treatment with an aqueous extract of D. moldavica causes sedative effects
364
such as prolonged sleeping time, sedation, reduced locomotor activity, and motor
365
coordination impairment in mice (Martinez-Vazquez et al., 2012). We cannot rule out that the
366
differences between the results of our study and the aforementioned study are due to
367
differences in the method of administration or extract preparation. Furthermore, a previous
368
study found that a total flavonoid extract of D. moldavica prevents learning and memory
369
deficits without causing motor impairments in APP/PS1 transgenic mice (Liu et al., 2018),
370
which supports our results. Collectively, these data indicate that D. moldavica may be a
371
potential agent for ameliorating cognitive dysfunction. 16
372
Previous phytochemical studies have revealed that oleanolic acid, rosmarinic acid,
373
chlorogenic acid, and apigenin are the major flavonoid compounds of D. moldavica
374
(Dastmalchi et al., 2007; Li et al., 2016). We also observed that EEDM contains rosmarinic
375
acid (31.24 ± 0.03 mg/g extract) and oleanolic acid (38.69 ± 0.26 mg/g extract). Mounting
376
evidence suggests that oleanolic acid ameliorates β-amyloid or scopolamine-induced
377
cognitive impairment through the activation of the TrkB-BDNF signaling cascade (Jeon et al.,
378
2017; Wang et al., 2018). Further, rosmarinic acid exhibits protective effects against Aβ-
379
induced cognitive impairment in the CNS (Alkam et al., 2007). Hasanein and Mahtaj (2015)
380
also reported that rosmarinic acid has an ameliorative effect on scopolamine-induced learning
381
and memory impairment in rats model (Hasanein and Mahtaj, 2015). Chlorogenic acid has
382
been reported to have neuroprotective and anti-amnesic effects against scopolamine-induced
383
amnesia in mice (Kwon et al., 2010). Another study indicated that apigenin improves
384
cognitive dysfunction and neuroinflammation via the upregulation of the ERK/CREB
385
pathway in APP/PS1 transgenic AD mice (Zhao et al., 2013). Together, these results suggest
386
that the memory-ameliorating effect of EEDM against scopolamine-induced impairment may
387
be attributed to the presence of these compounds.
388
It is well known that the cholinergic neurotransmission system in the basal forebrain
389
plays a critical role in learning and memory. Cholinergic transmission is mainly inactivated
390
by acetylcholine hydrolysis through AChE enzyme activity, which is responsible for the
391
degradation of acetylcholine into acetate and choline in the synaptic cleft (Ballard et al.,
392
2005). Excessive AChE activity leads to a persistent acetylcholine shortage and cognitive
393
dysfunction (Pepeu and Giovannini, 2010). Therefore, many researchers have focused on
394
searching for substances that can improve cognitive performance through the inhibition of
395
AChE activity. We also tested if EEDM could serve this purpose, as a previous reported study
396
indicated that Dracocephalum multicaule inhibits AChE enzyme activity (Mandegary et al., 17
397
2014). However, our results showed that EEDM does not have inhibitory activity against
398
AChE in an in vitro assay. These results suggest that the memory-ameliorating effect of
399
EEDM is not related to the inhibition of the AChE enzyme.
400
CREB is one of the key signaling molecules involved in learning and memory.
401
CREB is a transcription factor that functions as a molecular switch to control synaptic
402
plasticity and memory formation (Alberini, 2009). Meanwhile, the activation of CREB is
403
mediated by phosphorylation at serine 133, which can be controlled by ERK, Akt (also
404
known as protein kinase B), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and
405
protein kinase A (PKA) (Deak et al., 1998; Du and Montminy, 1998; Sun et al., 1994). ERK
406
activation is also highly associated with the development of several forms of memory,
407
including recognition and spatial memory (Kim et al., 2012). Previous studies have
408
confirmed the relationship between the ERK-CREB signaling pathway and memory
409
processing, suggesting that the activation of the ERK-CREB pathway possibly promotes
410
memory function (Davis et al., 2000; Peng et al., 2010). Our results showed that EEDM
411
significantly increased the phosphorylation levels of ERK and CREB in the hippocampus, but
412
not in the cerebral cortex. Notably, EEDM did not show any significant effects on the
413
phosphorylation levels of Akt, CaMKII or PKA in the hippocampus or cerebral cortex (data
414
not shown). These data indicate that the memory-ameliorating effect of EEDM may be due to
415
the activation of the ERK-CREB signaling cascade in the hippocampus. A previous study also
416
found that the flavonoid extract of D. moldavica effectively protects neurons against Aβ
417
accumulation and memory impairment by ERK-CREB pathway activation (Liu et al., 2018).
418
In addition, numerous studies have reported that activation of the ERK-CREB pathway is
419
associated with the enhancement of cognitive function as a result of the major constituent of
420
EEDM, including apigenin and oleanolic acid (Yi et al., 2014; Zhao et al., 2013). Therefore,
421
we suggest that the ERK-CREB signaling pathway is key mediator of the memory18
422
ameliorating effects observed in this study.
423
In conclusion, we demonstrated that EEDM effectively ameliorates scopolamine-
424
induced memory impairment in mice as measured by the passive avoidance and Morris water
425
maze tasks. Furthermore, EEDM may attenuate memory impairment through the activation of
426
the ERK-CREB pathway. These results suggest that D. moldavica may be used as a potential
427
therapeutic
428
neurodegenerative diseases.
agent
for
treating
cognitive
impairment
associated
with
various
429 430
Acknowledgments
431
This study was supported by the National Research Foundation of Korea (NRF) grant
432
funded by the Ministry of Science and ICT (NRF-2017R1C1B5017445; NRF-
433
2017R1A5A2014768).
434 435
Author's contributions
436
The study was conceived and designed by J.H.R. and S.J.P. Behavioral studies were
437
conducted by P.D., H.J.B. and H.P. Immunoblotting assays were performed by H.J.B, S.K.
438
and J.W.C. EEDM sample was prepared and standardized by X.L., S.K. and D.H.K.. The
439
manuscript was written by P.D., J.H.R. and S.J.P.
440 441
Conflict of interests
442
The authors declare that there is no conflict of interest.
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24
and
aberrant
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558 559
Figure legends
560 561
Figure 1. HPLC analysis of EEDM and standard compounds with detector responses at 280
562
and 210 nm. Detector responses were as follows: (A) Rosmaric acid at 280 nm, (B) EEDM at
563
280 nm, (C) oleanolic acid at 210 nm, and (D) EEDM at 210 nm. EEDM, ethanolic extract of
564
D. moldavica; HPLC, high performance liquid chromatography.
565 566
Figure 2. Effects of EEDM on unimpaired control mice and mice with scopolamine-induced
567
memory impairment in the passive avoidance task. (A) Step-through latency and (B)
568
electrosensitivity in unimpaired control mice; (C) Step-through latency and (D)
569
electrosensitivity in scopolamine-induced amnesic mice. The data represent the means ±
570
SEM (n = 9 - 10 per group). *p < 0.05 versus the vehicle-treated controls, #p < 0.05 versus the
571
scopolamine-treated group. Con, control; DNZ, donepezil; EEDM, ethanolic extract of D.
572
moldavica.
573 574
Figure 3. Effects of EEDM on scopolamine-induced memory impairment in the Morris water
575
maze task. (A) Latencies during the training trial sessions, (B) swimming time spent in the
576
target quadrant during the probe trial session and (C) swimming velocity during the probe
577
trial session. The data represent the means ± SEM (n = 8 - 9 per group)
578
the vehicle-treated controls; #p < 0.05, ##p < 0.01 versus the scopolamine-treated group. DNZ,
579
donepezil; EEDM, ethanolic extract of D. moldavica.
***
p < 0.001 versus
580 581
Figure 4. Effects of EEDM and donepezil on acetylcholinesterase (AChE) activity in vitro.
582
AChE activity was measured using a colorimetric assay using acetylthiocholine iodide as a
583
synthetic substrate. The AChE activity of each sample was observed three times. EEDM, 25
584
ethanolic extract of D. moldavica.
585 586
Figure 5. Effects of EEDM on ERK and CREB signaling cascades in the hippocampus of
587
scopolamine-induced amnesic mice. The immunoreactivity of phosphorylated ERK (pERK)
588
and ERK (A) and phosphorylated CREB (pCREB) and CREB (B) in the hippocampus were
589
quantified. The data represent the mean ± SEM. (n = 3-4/group) #p < 0.05 versus the
590
scopolamine-treated group. DNZ, donepezil; EEDM, ethanolic extract of D. moldavica ; Sco,
591
scopolamine.
26