Effect of Salvia miltiorrhiza root extract on brain acetylcholinesterase and butyrylcholinesterase activities, their mRNA levels and memory evaluation in rats

Accepted Manuscript Effect of Salvia miltiorrhiza root extract on brain acetylcholinesterase and butyrylcholinesterase activities, their mRNA levels and memory evaluation in rats

Marcin Ozarowski, Przemyslaw L. Mikolajczak, Anna Piasecka, Radoslaw Kujawski, Joanna Bartkowiak-Wieczorek, Anna Bogacz, Michal Szulc, Ewa Kaminska, Malgorzata Kujawska, Agnieszka Gryszczynska, Piotr Kachlicki, Waldemar Buchwald, Andrzej Klejewski, Agnieszka Seremak- Mrozikiewicz PII: DOI: Reference:

S0031-9384(16)30938-6 doi: 10.1016/j.physbeh.2017.02.019 PHB 11685

To appear in:

Physiology & Behavior

Received date: Revised date: Accepted date:

17 October 2016 1 February 2017 16 February 2017

Please cite this article as: Marcin Ozarowski, Przemyslaw L. Mikolajczak, Anna Piasecka, Radoslaw Kujawski, Joanna Bartkowiak-Wieczorek, Anna Bogacz, Michal Szulc, Ewa Kaminska, Malgorzata Kujawska, Agnieszka Gryszczynska, Piotr Kachlicki, Waldemar Buchwald, Andrzej Klejewski, Agnieszka Seremak- Mrozikiewicz , Effect of Salvia miltiorrhiza root extract on brain acetylcholinesterase and butyrylcholinesterase activities, their mRNA levels and memory evaluation in rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phb(2017), doi: 10.1016/j.physbeh.2017.02.019

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ACCEPTED MANUSCRIPT Effect

of

Salvia

miltiorrhiza

root

extract

on

brain

acetylcholinesterase

and

butyrylcholinesterase activities, their mRNA levels and memory evaluation in rats Marcin Ozarowskia,b,*, Przemyslaw L. Mikolajczakb,c, Anna Piaseckad,e, Radoslaw Kujawskib,c, Joanna Bartkowiak-Wieczorekf, Anna Bogaczg, Michal Szulcc, Ewa Kaminskac, Malgorzata Kujawskah, Agnieszka Gryszczynskab, Piotr Kachlickie, Waldemar Buchwaldi,

Department of Pharmaceutical Botany and Plant Biotechnology, Poznan University of

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a

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Andrzej Klejewskij,k, Agnieszka Seremak- Mrozikiewiczb,l,ł

b

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Medical Sciences, Sw. Marii Magdaleny 14, Poznan, Poland ([email protected]) Department of Pharmacology and Phytochemistry, Institute of Natural Fibers and Medicinal

Department of Pharmacology, University of Medical Sciences, Rokietnicka 5a, 60-806

Poznan,

Poland

([email protected],

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c

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Plants, Wojska Polskiego 71b, 60-630 Poznan, Poland ([email protected])

[email protected],

[email protected], [email protected])

Institute of Bioorganic Chemistry of the Polish Academy of Sciences, Noskowskiego 12/14,

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d

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61-704 Poznan, Poland ([email protected]) e

Department of Pathogen Genetics and Plant Resistance, Metabolomics Team, Institute of

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Plant Genetics of the Polish Academy of Science, Strzeszynska 34, 60-479 Poznan, Poland ([email protected], [email protected]) f

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Department of Clinical Pharmacy and Biopharmacy, University of Medical Sciences, Sw.

Marii Magdaleny 14, 61-861 Poznan, Poland ([email protected]) g

Department of Stem Cells and Regenerative Medicine, Institute of Natural Fibres and

Medicinal Plants, Wojska Polskiego 71b, 60-630 Poznan, Poland ([email protected]) h

Department of Toxicology, University of Medical Sciences, Dojazd 30, 60-631 Poznan,

Poland ([email protected])

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ACCEPTED MANUSCRIPT i

Department of Botany, Breeding and Agricultural Technology for Medicinal Plants, Institute

of Natural Fibres and Medicinal Plants, Wojska Polskiego 71b, 60-630 Poznan, Poland ([email protected]) j

Department of Nursing, University of Medical Sciences, Smoluchowskiego 11, Poznan,

Poland ([email protected]) Department of Obstetrics and Women’s Diseases, University of Medical Sciences,

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k

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Smoluchowskiego 11, Poznan, Poland ([email protected]) l

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Division of Perinatology and Women’s Diseases, University of Medical Sciences, Polna 33,

60-535 Poznan, Poland ([email protected])

Laboratory of Molecular Biology, University of Medical Sciences, Polna 33, 60-535 Poznan,

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ł

Poland ([email protected])

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*Corresponding author: Dr. Marcin Ożarowski, Department of Pharmaceutical Botany and Plant Biotechnology, University of Medical Sciences, Sw. Marii Magdaleny 14,

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Poznan, Poland

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Tel.: +48 61 6687849, fax: +48 61 6687861, e-mail: [email protected]

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ACCEPTED MANUSCRIPT Keywords: Salvia miltiorrhiza, memory, cholinesterase, gene expression, rat Abstract Salvia miltiorrhiza (Lamiaceae), one of the most important and popular plants of traditional medicine of Asia, is used for the prevention and treatment of cardiovascular diseases and in central nervous system disturbances. The main aim of this study was to assess the influence of

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subchronic (28-fold) administration of Salvia miltiorrhiza root extract (SE, 200 mg/kg, p.o.)

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on behavioural activity and memory of rats and to evaluate the activities of cholinesterases

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(AChE and BuChE) and gene expression levels of AChE and BuChE as well as of betasecretase (BACE1) in the hippocampus and frontal cortex in vivo. Huperzine A (HU, 0.5

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mg/kg b.w., p.o.) served as a positive control substance, whereas scopolamine (0.5 mg/kg, i.p.) injection was used as a well-known model of memory impairment. The results showed

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that subchronic administration of SE led to an improvement of long-term memory of rats. Strong inhibition of AChE and BuChE mRNA transcription in the frontal cortex of rats

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treated with SE or HU was observed. The BACE1 transcript level was significantly

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decreased. AChE activity was statistically significantly inhibited in the frontal cortex and the hippocampus by SE (47% and 55%, respectively). Similar effects were observed in the case

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of HU. In summary, activity of SE provides evidence that the plant can be a source of drugs

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used in the treatment of Alzheimer disease.

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ACCEPTED MANUSCRIPT 1. Introduction The rapid increase in the incidence of Alzheimer’s disease (AD) creates an urgent need for interventions to prevent or slow down the neurodegenerative process. The multiple pathogenic pathways of AD are associated with changes in the cholinergic system of the central nervous system and with β-amyloid deposition in the brain. The discovery and

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development of new drugs is often focused on chemical compounds from natural sources, i.e.

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from plant extracts [1]. Very interesting raw plant material in this area is the root of Salvia

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miltiorrhiza (Lamiaceae) [2]. This plant is one of the most important and popular plants of traditional medicine of Asia [2] and is mentioned not only in the Chinese Pharmacopoeia [3]

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but also in the European Pharmacopoeia [4]. Roots and rhizomes of this plant include two groups of bioactive compounds. The first group consists of numerous diterpenes such as

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tanshinone I, tanshinone IIA, tanshinone IIB, cryptotanshinone, 15,16-dihydrotanshinone, isotanshinone and salvinorin A. The other group comprises polyphenols such as rosmarinic

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acid, caffeic acid, salvianolic acid, lithospermic and ursolic acid [5]. The root of Salvia

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miltiorrhiza is widely used, mainly for prevention and treatment of cardiovascular diseases such as coronary heart disease, angina pectoris, myocardial infarction, myocardial ischaemia

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and atherosclerosis [2], in central nervous system disturbances and for neuroprotection (particularly in various models of neurodegenerative diseases) [6]. Five major tanshinones

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derived from root of Salvia miltiorrhiza exerted neuroprotective effects in the hippocampal CA1 [7], and in particular tanshinone I exhibited various inhibitory abilities to prevent unseeded amyloid fibril formation and to disaggregate preformed amyloid fibrils [8]. Furthermore, it was found not only that cryptotanshinone reveals neuroprotective properties relying on attenuation of beta-amyloid deposition through upregulating alpha-secretase in vitro and in vivo [9] and task learning improvement in rats with scopolamine-induced amnesia [10] but also that salvianolic acid B may cause inhibition of beta-amyloid fibril formation and 4

ACCEPTED MANUSCRIPT protection against the memory impairments induced by scopolamine and Aβ(25-35) [6]. Moreover, extract of root of Salvia miltiorrhiza exerted anti-acetylcholinesterase activity in vivo [11], while dihydrotanshinone and cryptotanshinone showed inhibition of human acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities in vitro [10, 12]. Huperzine A (HU) is a sesquiterpene alkaloid isolated from Huperzia serrata (Thunb. Ex

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Murray) Trev. (Huperziaceae) [13]. HU has been found to be a highly selective, reversible,

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and potent AChE inhibitor [14]. HU is a promising drug for treatment of symptoms of AD

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[14]. Several studies have shown that HU exerts memory-enhancing effects on a range of behavioural models in animals [15], therefore we chose the compound as a positive control

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substance in this study. In comparison with our previous articles, there was also studied the added impact of huperzine A on mRNA BACE1 gene expression level in rat brain.

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So far, studies have not fully explained the molecular basis of action of the Salvia miltiorrhiza radix extract (SE) and HU in different parts of the brain of experimental animals with

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aberrant memory processes.

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The main aim of this study was to assess the influence of subchronic administration of SE on behavioural activity and memory of rats and to evaluate AChE and BuChE activities and gene

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expression levels of AChE and BuChE as well as beta-secretase (BACE1) in the hippocampus and frontal cortex in vivo.

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2. Materials and methods 2.1. Plant material

Raw plant material (Salvia miltiorrhiza Bunge, Lamiaceae, root) was obtained from field crops of the Institute of Natural Fibres and Medicinal Plants in Poznan (Plewiska), Poland, in June, 2009. The voucher specimen (no. 976) of the plant was deposited in the Herbarium of this institution. 2.2. Preparation of extract 5

ACCEPTED MANUSCRIPT The roots of SE (1063 g) were extracted using 50% ethanol by percolation (24 h) at room temperature (22±1°C) and filtered. Then, the extract was concentrated under vacuum to eliminate the ethanol content. The concentrated extract was frozen at -55ºC and lyophilised. The final product yielded 483 g of solid extract. 2.3. Metabolite identification with LC-MS

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Phytochemical analyses were performed using two complementary LC-MS systems. The first

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system, HPLC-DAD-MSn, consisted of an Agilent 1100 HPLC instrument with a diode-array detector (DAD) (Agilent, Palo Alto, CA, USA) with an XBridge C18 column (150×2.1 mm,

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3.5 μm particle size) and an Esquire 3000 ion trap mass spectrometer (Bruker Daltonics,

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Bremen, Germany). Chromatographic separations by HPLC were carried out using water acidified with 0.1% formic acid (solvent A) and acetonitrile (solvent B) with mobile phase

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flow of 0.2 ml/min in the following gradient: 0-25 min from 10% to 30% B, 25-46 min to 98% B, and maintenance of these conditions up to 51 min. At 52 min the system was returned

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to starting conditions, with re-equilibration for 5 min. The most important MS parameters

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were as follows: the ion source ESI voltage -4 kV or 4 kV, nebulization of nitrogen at a pressure of 30 psi at a gas flow rate 9 l/min, ion source temperature 310°C, skimmer 1: -10 V.

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The spectra were scanned in the range of 50-3000 m/z. The second system consisted of UPLC (Acquity UPLC System, Waters, Milford, USA)

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hyphenated to a Q Exactive hybrid MS/MS quadrupole-Orbitrap mass spectrometer. Chromatographic separation for this system were carried out using water acidified with 0.1% formic acid (solvent A) and acetonitrile (solvent B) with mobile phase flow of 0.4 ml/min in the following gradient: 0-5 min from 10% to 25% B, 5-13 min to 98% B and maintenance of these conditions up to 14.5 min. At 15 min the system was returned to starting conditions, with re-equilibration for 3 min. The Q Exactive spectrometer was operated with the following settings: HESI ion source voltage -3 kV or 3 kV; sheath gas flow 50 l/min; auxiliary gas flow 6

ACCEPTED MANUSCRIPT 13 l/min; ion source capillary temperature 250°C; auxiliary gas heater temperature 380°C. The CID MS/MS experiments were performed using collision energy 15 eV. The MS n (up to the MS5) and MS/MS spectra were recorded in negative and positive ion modes using a previously published approach [1]. The individual compounds were identified via comparison of the exact molecular masses, mass spectra and retention times to those of standard

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compounds, online available databases and literature data.

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2.4. HPLC quantitative analysis

Quantitative HPLC analysis was performed on Agilent 1100 HPLC system (Lichrospher 100

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RP18 column (125 x 4 mm, 5 μm, Merck). Analysis HPLC-DAD was carried out by a

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gradient elution with flow rate of 0.8 mL/min. The mobile phases were 100% acetonitrile (solution A) and 0.1% trifluoroacetic acid, TFA (solution B) with used linear gradient from

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5% A (0 min), 77.5% A (35 min). Quantification was carried out with the use of a photodiode array detector (DAD) at 250 nm. All calculations were performed by external

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standardization by measurement of the peak areas.

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2.5. Determination of total phenolic compounds in the extract Calculation of polyphenols expressed as gallic acid was done using Folin-Ciocalteu reagent

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with the spectrophotometric method according to Singleton and Rossi [16]. 2.6. Determination of total hydroxycinnamic acid derivatives

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Determination of total hydroxycinnamic acid (HCA) derivatives calculated on rosmarinic acid was performed according to the procedure described in European Pharmacopoeia 6th edition [17 16]. 2.7. Chemicals and drugs All reagents for HPLC analysis, scopolamine hydrobromide trihydrate (S) and reagents for biochemical analyses were purchased from Sigma-Aldrich (Poland). Other substances used in HPLC such as tanshinones and rosmarinic acid were obtained from LGC Standard (Poland). 7

ACCEPTED MANUSCRIPT Huperzine A (HU) was obtained from Enzo Life Sciences AG (Alexis Corporation, Biomibo Distribution, Poland). Chemicals for gene expression analysis were obtained from Roche Diagnostic and ALAB (Poland). All chemicals and drugs were ex tempore prepared on the day of the experiment. 2.8. Animals

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Experiments with rats were performed in accordance with Polish governmental regulations

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(Dz. U. 05.33.289) and with EU Directive 2010/63/EU for animal experiments. The study was

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conducted in accordance with ethical research guidelines on conscious animals, and the study protocol was approved by the Local Ethics Committee on the Use of Laboratory Animals in

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Poznan, Poland (64/2008). The experiments were performed on male six week-old Wistar rats housed at controlled room temperature (20 ± 0.2°C) and humidity (65-75%) under a 12h : 12h

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light-dark cycle (lights on at 7 a.m.). The animals were kept in groups of 8-10 each in light plastic cages (60 x 40 x 40 cm) and had free access to standard laboratory diet (Labofeed B

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pellets) and tap water.

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2.9. Treatments

The water-ethanol (1:1) extract from the roots of Salvia miltiorrhiza was administered

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intragastrically (p.o.) in a dose of 200 mg/kg b.w. (groups SE+H2O and SE+S) and HU in a dose of 0.5 mg/kg b.w. (p.o.) (groups: HU+H2O and HU+S) once a day for 28 consecutive

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days. On the last day, 30 min after the last dose of SE, or HU, scopolamine (S) was given intraperitoneally (i.p.) in a dose of 0.5 mg/kg b.w. Control groups were treated with 0.5% methylcellulose (MC), and water for injection (H2O) was used as a vehicle for S (groups MC+H2O and MC+S). SE was prepared ex tempore before administration and suspended in MC in concentration of 10 mg/mL. Treatment regiment was described in the Table 1. Animal experimental procedures were summarized at Figure 1.

8

ACCEPTED MANUSCRIPT Table 1. Dosing regimen

MC + H2O

MC + S

Dose of substances administered

Dose of substances administered

intragastrically (p.o.) once a day for

intraperitoneally (i.p.) on the last day,

28 consecutive days

30 min before tests

0.5% methylcellulose (MC)

Aqua pro inj. (H2O)

10 ml/kg m.c.

1 ml/kg b.w.

0.5% methylcellulose (MC)

Scopolamine (S)

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Groups

10 ml/kg m.c. huperzine A

Aqua pro inj. (H2O)

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HU + H2O

0.5 mg/kg b.w.

HU + S

1 ml/kg b.w.

huperzine A 0.5 mg/kg b.w. Extract of Salvia miltiorrhiza 200 mg/kg b.w.

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Extract of Salvia miltiorrhiza 200

0.5 mg/kg b.w. Aqua pro inj. (H2O) 1 ml/kg b.w. Scopolamine (S) 0.5 mg/kg b.w.

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mg/kg b.w.

Scopolamine (S)

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SE + S

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SE + H2O

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0.5 mg/kg b.w.

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ACCEPTED MANUSCRIPT Figure 1. Testing scheme

1-28 days

 MC + H2O / HU + H2O / SE + H2O

 MC / HU / SE + Aqua pro inj. (H2O) // MC + S / HU + S / SE + S  after 30 min - locomotor activity assessment

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28th day

th

 after 30 min - the motor coordination test

 MC + H2O / HU + H2O / SE + H2O

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30 day

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29th day

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 MC / HU / SE + Aqua pro inj. (H2O) // MC + S / HU + S / SE + S

 after 30 min - the object recognition test

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31th day

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 MC / HU / SE + Aqua pro inj. (H2O) // MC + S / HU + S / SE + S

 MC + H2O / HU + H2O / SE + H2O

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32th day

 after 30 min - the passive avoidance test

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33th day

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 MC / HU / SE + Aqua pro inj. (H2O) // MC + S / HU + S / SE + S

 decapitation of rats and collection of the hippocampus and frontal cortex to biochemical studies

2.10. Behavioural and memory tests 2.10.1. Measurement of locomotor activity

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ACCEPTED MANUSCRIPT Locomotor activity assessment was performed (Activity Cage, Ugo Basile, Italy) by placing the animals in the apparatus and recording their horizontal activity. The data obtained were expressed as signals corresponding to animal movements for 5 minutes, after previous 20 minutes of habituation to the activity meter cage. The locomotor activity was measured 30

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minutes after the administration of a single dose of S or the vehicle (H2O).

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2.10.2. Measurement of motor coordination

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Motor coordination was evaluated using the “chimney” test originally designed for mice [18]. Thirty minutes after S or vehicle injection, a rat was allowed to enter a glass laboratory

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cylinder, 500 mm long and 80 mm in diameter, placed on its side. When the animal reached the cylinder bottom, the position of the cylinder was rapidly changed from horizontal to

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vertical and a timer was started. The rat began to move backwards immediately. The timer was stopped after the rat left the cylinder and assumed a sitting posture on the top of the

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vessel. Motor impairment was assessed as the inability of rats to climb backwards up the tube

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of S or the vehicle.

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within 60 s. The test was performed after 30 min following the administration of a single dose

2.10.3. Memory assessment

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Memory was evaluated with the passive avoidance test and object recognition test. 2.10.3.1. Passive avoidance test The passive avoidance test was performed (Passive Avoidance System – step-through, Ugo Basile, Italy) as a model for long-term memory assessment in animals [19]. After 2 minutes of habituation to the dark compartment, a rat was placed in the illuminated compartment and allowed to enter the dark compartment. Two more approach trials were allowed on the following day with a two-minute interval between them. At the end of the 11

ACCEPTED MANUSCRIPT second trial, an unavoidable scrambled electric footshock (500 A, AC, 3 s) was delivered through the grid floor of the dark compartment (learning trial). Retention of the passive avoidance response (latency) was tested 24 h later by placing the animal in the illuminated compartment and measuring the latency in re-entering the dark compartment against the arbitrary maximum time of 180 s. The test was performed after 30 min following the

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administration of a single dose of S or the vehicle. The test was previously described by

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Ozarowski et al. [20].

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2.10.3.2. Object recognition test

The object recognition test was used as a model for short-term memory assessment in animals

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[21]. The object recognition task took place in a 40×60 cm open box surrounded by 40 cm high walls made of plywood with a frontal glass wall. All animals were submitted to a

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habituation session during which they were allowed to freely explore the open area for 5 min. No objects were placed in the box during the habituation trial. On the day of testing, the

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animals were given an additional 3 min re-habituation period prior to commencing the test. So

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the test was divided into three phases with two trials: acquisition (explored two identical objects (A1 and A2)), inter-trial interval of varying times (returned to the home cage for 30

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min) and retention (explored a familiar object (A*) that was a duplicate of those objects from the acquisition trial (to minimize olfactory cues) and a novel object (B) for a further 3 min).

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The exploration times (s) of all objects were recorded with a stopwatch for subsequent statistical analysis. The time measured as an exploration behaviour was used to calculate the memory discrimination index (OR) as reported by Blalock et al. [21]: OR = (B-A*)/(B+ A), where B was the time spent exploring the new object and A* was the time spent exploring the familiar object. A higher OR was considered to reflect a greater memory ability [21]. The test was performed 30 min following the administration of a single dose of S or the vehicle.

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ACCEPTED MANUSCRIPT 2.11. Acetylcholinesterase and butyrylcholinesterase activities assay in rat brain On the last day of the experiments, 60 min after the last dose of SE or HU, the animals were killed by decapitation, and the hippocampus and frontal cortex were collected from the brains of the rats. The tissue samples were then stored at -80°C until measurement of activity of cholinesterases or their mRNA gene expression.

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The activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were

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performed by modified Ellman’s spectrophotometric method according to Isomae et al. [22]. The activity of AChE and BuChE was determined by measuring the formation of the yellow

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anions obtained from the reaction between Ellman’s reagent and the thiocholine generated by

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the enzymatic hydrolysis of acetylthiocholine iodide (ATCh) and butyrylthiocholine (BTCh), respectively (sample 0.1 mL, PBS 0.8 mL, DTNB 0.1 mL, ATCh 0.20 mL and BTCh 0.20

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mL). The biochemical assay of AChE and BuChE in the homogenate of brain samples was

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expressed as μmol/min/mg protein using the spectrophotometric method (UV 412 nm).

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2.12. RNA isolation, reverse transcription reaction and mRNA level changes quantification Total RNA isolation from the rat brain tissue homogenates (frontal cortex, hippocampus) was

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carried out using TriPure Isolation Reagent (Roche) according to the manufacturer’s protocol using a modified Chomczynski and Sacchi method [23]. quantitative

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Two-step

real-time

PCR

(qRT-PCR)

was

used

to

evaluate

the

acetylcholinesterase (AChE), butyrylcholinesterase (BChE), alpha- and beta-secretases genes expression levels. This

relative quantification method, prepared in a volume of 10 μL

reaction mixture, was carried out using a LightCycler TM Instrument (Roche, Germany) and a LightCycler Fast Start DNA Master SYBR Green I kit (Roche Applied Science), according to manufacturer's instructions. Reaction mixture compositions are: (1) mixture for AChE and BuChE: 7.1 µl RNA free water, 0.4 µl MgCl2 (final 2 mM), 0.25 µl starter forward (final 13

ACCEPTED MANUSCRIPT 0.5µM), 0.25 µl starter reverse (final 0.5 µM), 1 µl LightCycler Fast-Start Reaction Mix SYBR Green I (10 x), 1 µl cDNA; (2) mixture for GAPDH, beta-secretase: 6.9 µl RNA free water, 0.6 µl MgCl2 (final 2.5 mM), 0.25 µl starter forward (final 0.5 µM), 0.25 µl starter reverse (final 0.5 µM), 1 µl LightCycler Fast-Start Reaction Mix SYBR Green I (10 x), 1 µl cDNA.

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Reaction conditions for all molecular targets are shown in Table 2:

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Table 2. Reaction conditions for AChE, BuChE, beta-secretase (BACE1) (cycles 45) and for

AChE Step

BuChE Time

Temp.

(°C )

(s)

(°C )

Initialization

95

600

Denaturation

95

8

Annealing

65

6

Extension/elongation

72

Final elongation

70

Time

BACE1

GAPDH

Temp.

Time

Temp.

Time

(s)

(°C )

(s)

(°C )

(s)

95

600

95

600

95

600

95

8

95

8

95

4

65

6

61

6

56

8

7

72

8

72

8

72

8

20

70

20

70

20

70

20

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Temp.

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Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cycles 30)

Primers sequences for all analyzed genes were designed and custom-designed using the

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Oligo 6.0 software (National Biosciences) and were verified by assessment of a single PCR product on agarose gel and by a single temperature dissociation peak (melting curve analysis) of each cDNA amplification product. A Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as a housekeeping gene (endogenous internal standard)

as a

reference and endogenous control. The comparison of the CT value of target genes with that of the endogenous control gene allowed the gene expression level of the target genes to be normalized to the amount of input cDNA. 14

ACCEPTED MANUSCRIPT The relative quantification for any given gene was expressed as a signal relative to the average signal value for the internal standard. RT-PCR was carried out in a reaction mixture (10 μL) with proper concentrations of all components, according to manufacturer's instructions. For each quantified gene, standard curves were prepared from a cDNA dilution, and generated from a minimum of four data points. Complementary DNA was quantified by

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comparison of the number of cycles required for amplification of unknown samples with

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those of the series of cDNA standard dilutions. All quantitative PCR reactions were repeated twice. The data were evaluated using the LightCycler Run 4.5 software package (Roche non-template control to detect potential

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Applied Science). Each PCR run included a

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contamination of reagents. The primers sequences are shown in Table 3: Table 3. The primers sequences used to quantitative real-time PCR (GAPDH –

Primer sequence GEN

Primer sequence Reverse (5’ → 3’)

CAGCAATACGTGAGCCTGAA TTCCAGTGCACCATGTAGGA

BuChE

TAGCACAGTGTGGCCTGTCT

AACE

GGAGAGTTGGCCCCACAGTT CCCCTGGGATTGGAGTTAAGA

BACE1

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AChE

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Forward (5’ → 3’)

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Glyceraldehyde 3-phosphate dehydrogenase)

TCTTCATGTTGCCACTCTGC

TCTCTGTCATCTGCCCACTG

GATGGTGAAGGTCGGTGTG

ATGAAGGGGTCGTTGATGG

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GAPDH

TTCCACTCTTGCTCCCTTTC

2.13. Statistical analysis All values were expressed as means ± SEM. The statistical comparison of results was carried out using one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test for detailed data analysis. The level of statistical significance was set at p < 0.05. 3. Results 15

ACCEPTED MANUSCRIPT 3.1. Behavioural and memory tests 3.1.1. Locomotor activity A one-way ANOVA analysis revealed significant differences in the locomotor activity of rats, expressed as their horizontal spontaneous activity (F(5,69) = 6.91; p<0.001; Table 4). Detailed post-hoc analysis showed that SE+H2O statistically significantly diminished the

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locomotor activity of rats by 40% (spontaneous activity), in contrast to HU+H2O, which did

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enhance this activity as compared to the control group (MC+H2O). Stimulating significant

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effects in the locomotor activity of control rats were observed after an acute S injection (MC+S vs. MC+H2O, p<0.05), whereas SE+S and HU+S did not significantly affect this

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parameter.

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ACCEPTED MANUSCRIPT Table 4. Effects of multiple treatment (28x) of Salvia miltiorrhiza extract on behavioural activities and memory assessment in rats Groups

Locomotor

Motor coordination Long-term memory Short-term

activity

memory

spontaneous

Passive avoidance

Object

test [latency]

recognition test

activity [s]

after 24 h

impulses

[s]

17±3

54±15

0.40±0.06

(19)

(18)

(19)

(18)

526±49*

32±5*

(18)

(18)

515±32*

15±2

(9)

(8)

597±76

28±7

(9)

(8)

HU+H2O

HU+S

SE+H2O

240±38* (10) 590±69 (10)

CE

SE+S

13±3*

0.33±0.06

(19)

(18)

161±13**

0.38±0.08

(9)

(8)

45±16

0.22±0.06

(9)

(8)

22±4

140±26**

0.13±0.09*

(10)

(7)

(10)

41±6

37±18

0.25±0.10

(10)

(10)

(10)

PT E

MC+S

D

n

NU

401±24

MA

MC+H2O

SC

/5 min]

ratio ORB

RI

[number of

PT

Exit timeA

Horizontal

AC

n – number of rats (in brackets) values expressed as mean  SEM SE – extract from Salvia miltiorrhiza roots (200 mg/kg, p.o.) HU – huperzine A in a dose of 0.5 mg/kg, p.o. S – scopolamine in a dose of 0.5 mg/kg, i.p. A – exit time in the “chimney” test

17

ACCEPTED MANUSCRIPT B – ratio OR = (B-A*) / (B+A*), where: B is the time spent exploring the novel object (session II) ; A* is the time spent exploring the familiar object during session II (for details, see Materials and Methods) **,* – statistical difference vs. control (MC+ H2O), p < 0.01, p < 0.05, respectively 3.1.2. Motor coordination

PT

A one-way ANOVA analysis revealed significant differences in motor coordination of rats,

RI

expressed as their exit time from the cylinder (F(5,66) = 3.96; p < 0.05; Table 4). Detailed

SC

analysis showed that the acute S injection caused prolongation of exit time (MC+S vs. MC+H2O, p<0.05), whereas multiple administration of SE+H2O and HU +H2O or SE+S and

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HU +S did not significantly affect this parameter. 3.1.3. Long-term memory

MA

A one-way ANOVA analysis revealed significant differences in long-term memory after using the passive avoidance test (F(5,65) = 14.3; p=0.000, Table 4). The strongest effect

D

leading to an improvement of this parameter was caused by SE and HU as compared to

PT E

control animals (SE+H2O vs. MC+H2O, p < 0.01; HU+H2O vs. MC+H2O, p < 0.01). However, it was observed that S administration to rats significantly decreased the latency of

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the passive avoidance task (MC+S vs. MC+H2O, p < 0.05), but SE+S and HU+S did not significantly affect the long-term memory.

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3.1.4. Short-term memory

The results of the object recognition test revealed no significant differences in the short-term memory of rats (ANOVA F(5,66) = 1.88; p = 0.109) (Table 4). However, detailed analysis showed that only multiple SE administration impaired this memory in rats (SE+H2O vs. MC+H2O, p < 0.05). Whereas multiple administration of HU+H2O or SE+S and HU+S did not significantly affect the parameter. 3.2. Acetylcholinesterase and butyrylcholinesterase activities in rat brain 18

ACCEPTED MANUSCRIPT The results of the AChE activity showed significant differences both in the cortex (ANOVA F(2, 22) = 8.87; p = 0.002) and in the hippocampus of rats (ANOVA F(2,21) = 9.71; p = 0.001). The presence of SE extract caused statistically significant inhibition of AChE activity, by 47% (p < 0.01)) in the frontal cortex and by 55% (p < 0.01) in the hippocampus (Table 5). Similarly, HU exerted a strong inhibitory action in both regions of rat brain. In these regions

PT

BuChE activities were also slightly decreased but insignificantly (ANOVA: F(2,22) = 0.339;

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p = 0.716 and F(2,22) = 0.166; p = 0.848, respectively) (Table 5).

SC

3.3. mRNA AChE and BuChE gene expression levels in rat brain

Analysis (RT-PCR) of mRNA AChE and BuChE expression in rat brain homogenates showed

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significant differences in the cortex of rats (ANOVA: F(2, 23) = 7.48; p = 0.003 and F(2,20) = 5.93; p = 0.009, respectively). It was found that the SE resulted in a statistically significant

MA

decrease of relative expression levels of both mRNA AChE (by 41%, p < 0.01) and BuChE (by 48%, p < 0.05) (Table 5). Similarly, HU administration significantly lowered the

D

expression of AChE and BuChE in the cortex (by 44%, p < 0.01 and 58%, p < 0.01,

PT E

respectively). In contrast, in the hippocampus the results did not show significant differences for mRNA AChE or BuChE expression (ANOVA: (F(2,26) = 2.25; p = 0.125 and F(2,25) =

AC

CE

0.807; p = 0.457, respectively) (Table 5).

19

ACCEPTED MANUSCRIPT Table 5. Effect of extract from leaves of Salvia miltiorrhiza (200 mg/kg, p.o.) after its subchronic treatment on AChE and BuChE activities, and on mRNA AChE, BuChE or BACE1 gene expression in rat brain. BuChE

[nmol

[nmol

ATCh/min/mg

BuTCh/min/

protein]

mg protein]

Hipp.

Corte

Hip

Corte

Hipp.

x

p.

x

[%]

[%] 439±76

2O

7

HU

189±1

229±15

+H2O

5*

*

SE+

192±2

199±18

H2O

9*

**

65±1

53±

100

1

8

12

57±7

51±

56±6

4

**

55±9

mRNA

BuChE

BACE1

Corte

Hipp.

x

Corte

Hipp.

x

[%]

[%]

[%]

[%]

100

100

100

100

11

18

11

16

85±5

42±9

102±

62±6

**

11

*

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363±4

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MC+H

mRNA

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Cortex

mRNA AChE

PT

AChE

SC

Groups

1008

98±4

48±

59±8

108±

52±8

120±

62±7

133±6

6

**

7

*

14

*

**

D

Value expressed as mean ± SEM (n = 8-10)

Hipp. – hippocampus

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Control group (MC+H2O) defined as 100%

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AChE – acetylcholinesterase

BuChE – butyrylcholinesterase

AC

BuTCh – butyrylthiocholine BACE1 – beta-secretase

HU – in a dose of 0.5 mg/kg, p.o. SE – extract from roots of Salvia miltiorrhiza **,* – statistically significant difference vs. control (MC+ H2O), p < 0.01 or p < 0.05, respectively

20

ACCEPTED MANUSCRIPT 3.4. mRNA BACE1 gene expression level in rat brain Statistical analysis revealed significant differences in mRNA BACE1 expression in the cortex (ANOVA F(2,23) = 4.47; p = 0.023). Both SE and HU treatment caused a decrease of their mRNA expression in this region of rat brain (by 48%, p < 0.05) (Table 5). In the hippocampus there was also a significant difference between mRNA BACE1 expression levels (ANOVA

PT

F(2,23) = 9.50; p = 0.001), but the effect was caused only by SE administration, showing a

SC

RI

33% increase (p < 0.01) compared with control values.

3.5. Phytochemical profile of ethanol extract of Salvia miltiorrhiza roots

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The major compounds in hydro-ethanolic Salvia miltiorrhiza root extract established by HPLC-DAD were found to be tanshinones (1.07%) such as tanshinone I (0.40%), tanshinone

MA

IIA (0.15%), cryptotanshinone (0.33%) and dihydrotanshinone (0.19%). Moreover, rosmarinic acid (0.15%) was determined in the extract. The total polyphenol

D

content of SE was determined using the Folin–Ciocalteu assay. The extract showed 12.89%

HPLC was 6.92%.

PT E

gallic acid. The total content of hydroxycinnamic derivatives expressed as rosmarinic acid by

CE

Phytochemical analyses were performed using two complementary LC-MS systems (HPLCDAD-MSn, UPLC hyphenated to Q-Exactive hybrid MS/MS quadrupole-Orbitrap mass

AC

spectrometer) which allowed the identification of 32 secondary metabolites in hydro-ethanolic Salvia miltiorrhiza root extract. Among them two structural groups may be distinguished. The first group included polyphenolics in the form of monomers, dimers, trimers and tetramers of hydroxycinnamic acids. Only two metabolites (feruloyl-di-glucoside, and protocatechuic acid) were not derivatives of caffeic acid, which is a building block for a variety of condensation products of caffeic acid and 3,4-dihydroxy-phenyl lactic acid (danshensu). The second group

21

ACCEPTED MANUSCRIPT represented diterpene tanshinones, mainly from two skeleton forms: 20-norabietanes and 19,20-dinorabietanes.

4. Discussion The purpose of our study was to investigate the influence of subchronic (28-fold)

PT

administration of standardized 50% EtOH extract of S. miltiorrhiza (200 mg/kg, p.o.) and HU

RI

(0.5 mg/kg b.w., p.o.) on scopolamine-impaired short-term and long-term memory. The

SC

results were compared with the activity of cholinesterases (AChE and BuChE) as well as with AChE and BuChE gene expression levels in the cortex and hippocampus of the rat brain.

NU

In this study, scopolamine (S) injection was used as a well-known model of memory impairment [20]. However, we found a notable enhancement of the locomotor activity in rats

MA

subjected to S. It is probably due to the fact that S in the dose used in this study does not act as a CNS depressant and stimulates exploratory behaviour in rodents by muscarinic

D

antagonism, facilitating the release of an excitatory neurotransmitter, i.e. acetylcholine (turn-

PT E

over effect) [24]. Moreover, S treatment led to reduced motor coordination. However, it is also claimed that S demonstrates a lack of correlation between motor skills and learning

CE

abilities [25]. Therefore, the question whether the impairment of motor coordination and simultaneous increase of locomotor activity of rats caused by S can affect the memory

AC

remains open. It should also be stressed that SE administration changed the effect of S on locomotor activity, whereas the presence of SE resulted in a reduction of that activity in non S-injected rats. Therefore it can be stated that in this way SE sometimes exerts the ascribed sedative activity [26]. However, there were no effects of SE on exit time in the “chimney test” in either S-treated or S-non-treated animals, so changes of motor coordination during the presence of SE should be excluded.

22

ACCEPTED MANUSCRIPT The presence of SE in animals treated with or without S showed a stimulating effect on long-term memory vs. the control. The influence of SE+ S (induction by 208%) (p>0.05) was stronger than in animals treated with SE only (174% induction). In contrast, no significant improvement of the short-term memory was observed after SE administration to rats. There is some evidence from other studies supporting our results, although the results cited below have

PT

sometimes been conducted in different experimental conditions. Moreover, most of the

RI

pharmacological studies, apart from our study, were focused on the assessment of the activity

SC

of individual chemical compounds rather than the crude extracts of S. miltiorrhiza root. Lee et al. [6] (2013) observed that subchronic administration of salvianolic acid B (7 days, 10 mg/kg

NU

b.w.) ameliorated Ab25–35 peptide-induced cognitive dysfunction in mice because it significantly increased the mean step-through latency in the passive avoidance task compared

MA

with the Ab25–35 peptide-treated group. Salvianolic acid B also rescued impairment in the cholinergic neurotransmitter system [11]. Other studies have also shown that tanshinone I (2

D

or 4 mg/kg, p.o.), tanshinone IIA (10 or 20 mg/kg, p.o.), cryptotanshinone (10 mg/kg, p.o.)

PT E

and 15,16-dihydrotanshinone I (2 or 4 mg/kg, p.o.) have the ability to ameliorate memory deficits induced by scopolamine (1 mg/kg, i.p.) in a mouse model (the passive avoidance

CE

task), by enhancing cholinergic signalling [27]. Moreover, tanshinone I (4 mg/kg, p.o.) and tanshinone IIA (10 or 20 mg/ kg, p.o.) significantly attenuated diazepam-induced reductions

AC

in step-through latency via cellular kinase signalling [27]. In another study, the task-learning ability of scopolamine-treated rats was significantly reversed by cryptotanshinone (5 mg/kg) [10]. A potentially significant neuroprotective potential was also supported by the results from other studies showing that extracts from roots of S. miltiorrhiza improved blood flow and resolution of blood stasis [28].

23

ACCEPTED MANUSCRIPT Concerning the effect of HU on memory, for example Ohba et al. [15] observed in scopolamine-induced cognitive impairment of mice that H. serrata extract (30 mg/kg/day) which contained 0.5% huperzine administered for 7 days per os inhibited AChE but not BuChE. Moreover, this extract ameliorated cognitive function in mice (the Y-maze and passive avoidance tests).

PT

These observations may lead to the conclusion that SE can be taken into consideration

RI

in treatment not only of cerebrovascular diseases but also vascular dementia, defined as loss

SC

of cognitive function resulting from ischaemic, haemorrhagic brain lesions or hypoperfusion, due to cerebrovascular disease or cardiovascular pathology [29], and mixed dementia, in

NU

which Alzheimer’s disease and vascular dementia occur at the same time for the majority of cases worldwide. Moreover, the close interrelationship between Alzheimer’s disease and

MA

cerebrovascular diseases suggests the necessity of the maintenance of cerebrovascular integrity, which may herald a new generation of dementia treatment strategies [30]. In this

D

context, SE may be an interesting option in phytotherapy because reducing cerebrovascular

Alzheimer’s disease.

PT E

disease may offer long-term preventative measures for both vascular dementia and

CE

The results from our study demonstrate that subchronic administration of SE led to an improvement of long-term memory of rats, which can be partially explained by its inhibitory

AC

action on AChE activity in rats’ frontal cortex and hippocampus. Indeed, AChE activity was statistically significantly inhibited in the frontal cortex by 47% and by 55% in the hippocampus of rat brain, which may suggest its crucial neuroprotective property in this matter. These results are in line with those of Kim et al. [27] in which that cryptotanshinone and 15,16-dihydrotanshinone I (pharmacologically active compounds present in SE) were found to have an inhibitory effect on acetylcholinesterase in vitro in mouse brain 24

ACCEPTED MANUSCRIPT homogenates with IC(50) values of 82 and 25 μM, respectively. In another study, Wong et al. [10] demonstrated that cryptotanshinone is an inhibitor of both human acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with IC(50) values of 4.09 and 6.38 μM, respectively. A further study of Wong et al. [12] also showed that 15,16- cryptotanshinone and dihydrotanshinone are mixed non-competitive inhibitors for recombinant human AChE

PT

(4.67 and 0.89 μM, respectively) and uncompetitive inhibitors for BChE (6.66 and 5.51 μM,

RI

respectively) in vitro. Zhou et al. [11] observed that not only selected tanshinones but also

SC

ethanol extract of Salvia miltiorrhiza radix had a remarkable inhibitory effect on acetylcholinesterase in vitro in rat brain homogenates.

NU

The observed results from the behavioural experiments and changes in AChE and BuChE enzyme activities are at least partially reflected in the transcriptional profile changes

MA

of studied AChE, BuChE and BACE1 mRNAs, particularly in the frontal cortex. In our study strong inhibition of AChE and BuChE mRNA transcription in the frontal

D

cortex of animals treated with SE was found, and the BACE1 transcript level was

PT E

significantly decreased. On the other hand, there were no significant changes after SE administration in the hippocampus, and even SE showed an increasing effect on BACE1

CE

mRNA expression.

To date, there is a lack of published results of studies attempting to clarify the

AC

potential differences in the transcriptional profiles of AChE and BuChE genes and activities of their proteins in rat brain under the influence of SE. The observed differences in the relative mRNA expression levels of AChE and BuChE between the cortex and hippocampus in studied rats in comparison to their activity can be explained by their diverse location, structure and function as well as by their quite complicated and not fully understood molecular mechanisms regulating their activities. The difference in AChE, BuChE mRNA transcription may be due to the location of the AChE enzyme and the prevalence of its 25

ACCEPTED MANUSCRIPT different molecular forms that may be responsible for this protein’s quantitative, spatial and temporal variations [31]. The present study only assessed the overall total pool of mRNA and the overall level of activity of AChE enzyme. Consequently, further research will be necessary to assess the activity and separate expression of mRNA of different isoforms of the enzyme. A complex understanding of the molecular basis of changes in the transcription

PT

profile of analyzed genes also requires identification and assessment of the degree of

RI

involvement of transcription and epigenetic factors regulating the synthesis of studied

SC

mRNAs.

In summary, the results of our comparative study in an animal model on activity of

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extract from root of Salvia miltiorrhiza provide evidence that the plant can be source of drugs

MA

used in treatment of AD.

5. Conclusion

D

The subchronic administration of SE led to an improvement of long-term memory of rats,

PT E

which can be partially explained by its inhibitory action on AChE activity in rats’ frontal cortex and hippocampus. It seems that the SE activity represents a possible option for

CE

preventive treatment against some neurodegenerative diseases. Acknowledgements

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This study was carried out within the framework of research project no. N 405417836 financed by the Polish Ministry of Science and Higher Education (National Board of Sciences, Poland).

Conflict of interest The authors declare no conflict of interest with any financial organization regarding the material discussed in the manuscript. 26

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ACCEPTED MANUSCRIPT

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Graphical abstract

Highlights

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CE

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MA

NU

SC

We investigated the influence of Salvia miltiorrhiza extract on activities of rats We investigated the influence of huperzine A on activities of rats We evaluated the cholinesterases activity in the hippocampus and frontal cortex We evaluated the gene expression levels of cholinesterases and beta-secretase We evaluated phytochemical composition of Salvia miltiorrhiza extract

31