Ethanol extract of Astragali Radix and Salviae Miltiorrhizae Radix, Myelophil, exerts anti-amnesic effect in a mouse model of scopolamine-induced memory deficits.

Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Research Paper

Ethanol extract of Astragali Radix and Salviae Miltiorrhizae Radix, Myelophil, exerts anti-amnesic effect in a mouse model of scopolamine-induced memory deficits Jin-Seok Lee a, Hyeong-Geug Kim a, Jong-Min Han a, Dong-Woon Kim b, Min-Hee Yi b, Seung-Wan Son c, Young-Ae Kim a, Jong-Suk Lee d, Min-Kyeong Choi a, Chang-Gue Son a,n a

Liver and Immunology Research Center, Oriental Medical Collage of Daejeon University, 22-5 Daehung-dong, Jung-gu, Daejeon 301-724, Republic of Korea Department of Anatomy, Brain Research Institute, Chungnam National University School of Medicine, Daejeon, Republic of Korea Department of Biomedical Engineering, College of Health Science, Korea University, Seongbuk-Gu, Seoul 136-703, Republic of Korea d GyeongGi Bio-Center, GSTEP, 864-1 Iui-dong, Yeongtong-gu, Suwon, Gyeonggi-do, Republic of Korea b c

art ic l e i nf o Article history: Received 18 July 2013 Received in revised form 20 November 2013 Accepted 18 March 2014 Keywords: Scopolamine Acetylcholinesterase Extracellular signal-regulated kinase Memory Oxidative stress Antioxidant

a b s t r a c t Ethnopharmacological relevance: Myelophil, a combination of extracts taken from Astragali Radix and Salviae Miltiorrhizae Radix, is a traditional Chinese medicine used for the treatment of chronic fatigueQ3 associated disorders. Here we examined the ability of Myelophil to alleviate memory impairment in a mouse model. We aimed to investigate whether Myelophil has the pharmacological effects on memory deficits associated with brain dysfunctions using an animal model. Materials and methods: Ten week-old male C57BL/6N mice were pretreated with Myelophil (50, 100, or 200 mg/kg), or tacrine (10 mg/kg) for 7 days, and then intraperitoneally injected with scopolamine (1 mg/kg). Memory-related behaviors were evaluated using the Morris water maze for 5 days. Levels of biomarkers of oxidative stress, antioxidant activity, acetylcholinesterase (AChE) activity, and extracellular signal-regulated kinase (ERK) were measured in brain tissues. Results: Scopolamine treatment increased the escape latency time and shortened time spent in the target quadrant; these effects were ameliorated by pretreatment with Myelophil. Scopolamine-induced changes in reactive oxygen species (ROS), malondialehyde (MDA), and AChE activity were significantly attenuated in mice pretreated with Myelophil. Recovery of antioxidant capacities, including total glutathione (GSH) content, and the activities of GSH-reductase, GSH-S-transferase, and catalase was also evident in Myelophil-treated mice. The strongest effects were seen for ERK and muscarinic acetylcholine receptor 1 (mAChR1) at both the protein and gene expression levels, with significant amelioration of expression levels in the Myelophil pretreatment group. Conclusions: These results suggest that Myelophil confers anti-amnesic properties in a mouse model of memory impairment, driven in part by the modulation of cholinergic activity. & 2014 Published by Elsevier Ireland Ltd.

1. Introduction As populations age, degenerative disorders of the central nervous system (CNS) including Alzheimer's disease, Parkinson's disease, and Huntington's disease have emerged as significant public health care challenges (Ross and Poirier, 2004). These diseases are associated with serious physical and social problems characterized by motor disturbances and psychological complications involving memory loss, concentration problems, and depression (Martin, 1999). Although the etiology and pathogenesis of

n

Corresponding author . Tel.: þ8242 257 6397; fax: þ8242 257 6398. E-mail address: [email protected] (C.-G. Son).

these disorders are not fully understood, the most important processes appear to be driven by degenerative changes in the cholinergic neurons leading to cholinergic neurotransmitter dysfunction (Aarsland et al., 2004; Bartus et al., 1982). Oxidative stress is an important factor in the pathophysiology of neurodegenerative disorders (Jomova et al., 2010). Previous reports have shown the increased expression of biomarkers associated with oxidative stress such as malondialdehyde (MDA) and reactive oxygen species (ROS) in brain cells and other neural tissue (El-Sherbiny et al., 2003). Brain tissue is particularly susceptible to oxidative stress due to its high consumption of oxygen, low antioxidant capacity, and relatively high content of iron and polyunsaturated fatty acids (Choi, 1993; Halliwell, 1992). Within the brain, these areas most vulnerable to oxidative stress include the basal forebrain and amygdala. As these

http://dx.doi.org/10.1016/j.jep.2014.03.048 0378-8741/& 2014 Published by Elsevier Ireland Ltd.

Please cite this article as: Lee, J.-S., et al., Ethanol extract of Astragali Radix and Salviae Miltiorrhizae Radix, Myelophil, exerts antiamnesic effect in a mouse model.... Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.03.048i

J.-S. Lee et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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regions are important for brain-specific functions such as cognition and memory (Mattson et al., 1999), damage to these areas can have significant neurological effects. Astragalus and salvia root (Radix Astragali and Radix Salviae Miltiorrhizae, respectively) are widely used in traditional Chinese medicine for the treatment of a wide array of ailments. Our previous studies have shown that Myelophil, an ethanol extract of Radix Astragali and Radix Salviae Miltiorrhizae, ameliorates oxidative damage in the brains of mice using a chronic restraint stress model of disease (Lee et al., 2012). Myelophil also conferred hepatoprotective effects against acute stress-induced liver injury (Kim et al., 2012), and anti-fatigue effects in a clinical study of chronic fatigue syndrome (Cho et al., 2009). Taken together, these findings suggest a common theme in which Myelophil confers protection against a variety of disorders by means of its potent antioxidant effects. Here we expand upon this hypothesis, and examine whether Myelophil could be used to treat neurodegenerative disorders associated with oxidative stress. We investigated the pharmacological effects of Myelophil on learning and memory in a scopolamine-induced memory impairment model of disease, and examined potential mechanisms of action underlying these effects.

diammonium salt (ABTS), 4-amino-3-hydrazino-5-mercapto-1,2,4triazole (Purpald), 1-chloro-2,4-dinitrobenzene (CDNB), N,Ndiethyl-p-phenylendiamine (DEPPD), 2,4-dinitrophenylhydrazine (DNPH), 5,50 -dithio-bis-2-nitrobenzoic acid (DTNB), ferrous sulfate, potassium phosphate, reduced glutathione (GSH), myoglobin, glutathione reductase (GSH-Rd), L-glutathione oxidized disodium salt (GSSG), guanidine hydrochloride, lipopolysaccharide (LPS), potassium phosphate reduced form of β-nicotinamide adenine dinucleotide phosphate (β-NADPH), scopolamine hydrobromide, 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate (tacrine), trichloroacetic acid (TCA), 1,1,3,3-tetraethoxypropane (TEP), and tert-butyl hydroperoxide. Others had been used as follows; Western blot antibodies such as extracellular signal-regulated kinase (ERK), phosphor-ERK, beta-actin, and secondary HRP-conjugated (Cell Signaling; Danvers, MA; and Santa Cruz Biotechnology; Santa Cruz, CA) Thiobarbituric acid (TBA; Lancaster Co.; Lancashire, England), hydrogen peroxide, (Junsei Chemical Co., Ltd.; Tokyo, Japan), nbutanol (J.T.Baker; Mexico City, Mexico), 1 M Tris–HCl solution (pH 7.4) and 500 mM ethylene diamine tetraacetic acid (EDTA) solution (pH 8.0; Bioneer; Daejeon, Republic of Korea). 2.4. Animals and experimental design

2. Materials and methods 2.1. Materials Astragali Radix (Astragalus membranaceus Bunge, cultivated in Jecheon, South Korea Ser. NO. 20101106-JC-HG) and Salviae Miltiorrhizae Radix (Salvia miltiorrhiza Bunge, cultivated in Hebei, China; Ser. NO. 20110302-CHN-DS) were purchased from an Eastern medicine company (Jeong-Seong Drugstore, Daejeon, Korea). The information of Myelophil is summarized in Table 1. 2.2. Preparation and fingerprinting of Myelophil Equal proportions of Astragali Radix and Salviae Miltiorrhizae Radix were extracted using 30% ethanol, and formulated into Myelophil (Kyung-Bang Pharmaceutical Company; Seoul, Korea) as per Korean monographs. Totally, 100 kg of herbal plants were extracted (each herb for 50 kg) and obtained 20.52 kg of product. The final Myelophil product [yield: 20.52% (w/w)] was stored for future use (VS No: KBMyelophil-2011-01). The Myelophil composition was confirmed by fingerprinting using reference compounds (astragaloside IV and formononetin for Astragali Radix and salvianolic acid B and rosmarinic acid for Salviae Miltiorrhizae Radix respectively) by ultra highperformance liquid chromatography (UHPLC) (Thermo Scientific, San Jose, CA, USA) as previously reported (Lee et al., 2012). The representative sample chromatogram and quantitative analysis were displayed in Fig. 1A and B.

Forty-eight specific pathogen-free C57BL/6N male mice (10 weeks old; 24–27 g, from Koatech; Gyeonggido, Korea) were used for the study. The protocol was approved by the Institutional Animal Care and Use Committee of Daejeon University (DJUARB 2012-015) and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH). The mice had ad libitum access to food pellets (Cargill Agri Furina; Gyeonggido, Korea) and water housed at room maintaining 2372 1C with 12 h:12 h light–dark cycle. After acclimatization for 1 week, the mice were randomly divided into six groups (each with n ¼ 8): naïve (pretreatment with distilled water for 7 days and saline injection once), control (pretreatment with distilled water for 7 days and scopolamine injection once), Myelophil (pretreatment with 50, 100, or 200 mg/kg Myelophil for 7 days and scopolamine injection once), and positive control (pretreatment with 10 mg/kg tacrine for 7 days and scopolamine injection once). The dose of Myelophil for current experiment was decided based on clinical use. In general, 60 kg of adult takes 1 to 2 g of Myelophil daily. Considering conversion factor between mice and human, we administered 200 mg/kg dosage of Myelophil daily to mice. The Myelophil and tacrine were dissolved in distilled water and administered orally for 7 days prior to scopolamine injection. The scopolamine was dissolved in 0.9% physiological saline (1 mg in 10 mL), and injected intraperitoneally (ip) to mice (100 μL/10 g of body weight) 30 min before behavioral testing in the Morris water maze. 2.5. Sample preparation

2.3. Chemicals and reagents The following reagents were obtained from Sigma (St. Louis, MO, USA); 2,20 -zino-bis (3-ethylbenzothiazoline-6-sulfonic acid)

All the mice used were sacrificed on the last day under mild ether anesthesia. Blood samples were collected from an abdominal vein, and the brains were immediately removed. The blood serum

Table 1 The herbal prescription of Myelophil. Herbal name

Scientific name

Local name

Place of origin

Relative amounts (kg)

Astragali Radix Salviae Miltiorrhizae Radix Final yield (%)

Astragalus membranaceus Bunge Salvia miltiorrhiza Bunge –

Huángqí Dan Shen –

South Korea (Jecheon) China (Hebei) –

50 50 20.52



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Fig. 1. UHPLC/MS chromatogram of Myelophil. (A) Myelophil and five standards were subjected to UHPLC analysis. (B) Four major compounds were quantitative analyzed for Myelophil.

was collected by centrifuging (4 1C, 3000 rpm for 15 min) and stored at 80 1C until biochemical assay. The remaining brain tissue, except cerebellum, was dissected and stored at 80 1C or in RNA later (Ambion; TX, USA) until use. 2.6. Determination of spatial memory using the Morris water maze task The Morris water maze task for spatial learning and memory testing was performed in circular pool (100-cm diameter 50-cm height) with circular acrylic platform (10-cm diameter 35-cm height) and a visual cue around the pool. The pool was filled with milk water (2271 1C) and divided into four equal quadrants. A platform was placed in one of the four quadrants approximately 1 cm below the surface as previously described (Harrison et al., 2008). Data were acquired via a video camera connected to corresponding software (Smart Junior, Panlab SL; Barcelona, Spain). Animals were pretreated with Myelophil for 7 days. Before behavioral testing, the animals were treated with scopolamine injection followed by 30 min of rest, and tested in the Morris water maze. Mice were placed on the platform for 10 s and then removed before the first test. For the first 4 testing days, the escape latency to platform and cumulative path-length were recorded during each acquisition trial. Finally, their behavior was recorded for 120 s after removing the platform, as a probe trial on the fifth day. During this trial, the time spent in the target quadrant was measured as an additional informative indicator of spatial learning and memory. 2.7. Determination of AChE in brain tissue Acetylcholinesterase (AChE) activity in brain tissue was determined using commercially available AChE activity assay kit (AAT

Bioquest; Sunnyvale, CA, USA) according to the manufacturer's protocol and resulting absorbance measured at 570 nm using UV spectrophotometer (Molecular Devices; Sunnyvale, CA, USA). 2.8. Western blot analysis Brain tissue was homogenized on ice using radioimmunoprecipitation assay (RIPA) buffer. Aliquots were mixed with equal amounts of protein, separated by 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 5% skim milk with advanced blocking reagent, the membranes were probed overnight at 4 1C with primary antibodies (ERK, p-ERK, and β-actin). The membranes were washed and incubated for 2 h at room temperature with horseradish peroxidase-conjugated anti-rabbit antibody. Western blots were revealed using an ECL advanced kit and quantified using the Image J 1.46 software (Rasband, WS, ImageJ; NIH, Bethesda, MD, http://rsb.info.nih.gov/ij). 2.9. Determination of ROS in serum and brain tissue and NO levels in brain tissue ROS levels in serum and brain tissue were determined as previously described (Hayashi et al., 2007). Briefly, 5 mL of standard solution or sample was mixed with 140 mL of 0.1 M sodium acetate buffer (pH 4.8) in 96-well plate and incubated at 37 1C for 5 min, 100 mL of (1:25) DEPPD (10 mM)/ferrous sulfate solution (4.37 μM) mixture was added to each well. Resulting absorbance was measured at 505 nm by UV spectrophotometer (Molecular Device). The level of ROS was calculated using H2O2 as standard and expressed as unit/mL.



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Levels of nitric oxide (NO) in brain tissue were determined using the Griess method (Green et al., 1982). Briefly, 100 mg of mashed brain tissue was homogenized in 2 mL of RIPA buffer. Then, 40 μL of homogenate was transferred to 96-well plate, and 160 μL of Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl) ethylenediamine hydrochloride, 2.5% H3PO4) was added and incubated at 37 1C for 20 min, resulting purple azo dye product was measured at 540 nm using UV spectrophotometer (Molecular Devices).

2.10. Determination of MDA in brain tissue Levels of lipid peroxidation (MDA) in brain tissue were measured using the thiobarbituric acid reactive substance (TBARS) method as previously described (Mihara and Uchiyama, 1978). Briefly, a 10% (w/v) brain tissue homogenate was prepared with ice cold 1.15% KCl, and 0.13 mL of homogenate was mixed with 0.08 mL of 1% phosphoric acid and 0.26 mL of 0.67% TBA solution. After incubating the mixture at 100 1C for 45 min, 1.03 mL of n-butanol was added. The mixture was then vortexed and centrifuged at 3000g for 15 min at 4 1C. Absorbance was measured at 535 nm and 520 nm using a spectrophotometer (Molecular Devices). The concentration of TBARS was calculated using prepared TEP as a standard and expressed as mmol/mg protein.

2.11. Determination of protein carbonyl contents in brain tissue Protein carbonyl contents in brain tissue were determined according to the manufacturer's protocol (Levine et al., 1994). Briefly, the brain tissue homogenate was prepared with cold phosphate buffer (50 mM, pH 6.7; containing 1 mM EDTA). Samples of 0.2 mL of homogenate were transferred to S# (sample) and C# (control) tubes separately. Then 800 μL of DNPH (10 mM) and 2.5 M HCl were added to the S# tube and the C# tube, respectively. Tubes were incubated in the dark at room temperature for 1 h while vortexing every 15 min before 1 mL of 20% TCA was added to each tube. After placing them on ice for 5 min, the tubes were centrifuged at 10,000g for 10 min at 4 1C. The supernatant was discarded, and 1 mL of 10% TCA was added. They were placed on ice for 5 min and then centrifuged at 10,000g for 10 min at 4 1C. The precipitates were suspended in 1 mL of an ethanol/ethyl acetate mixture (1:1) using a spatula and vortex. Then they were centrifuged at 10,000g for 10 min at 4 1C; the process was repeated two more times. The precipitates were re-suspended in 500 μL of guanidine hydrochloride by vortexing and were centrifuged at 10,000g for 10 min at 4 1C. Subsequently, 220 μL of supernatant was transferred to 96-well plates, and absorbance at 370 nm was measured using a spectrophotometer (Molecular Devices).

2.12. Determination of TAC in brain tissue Indicators of total antioxidant capacity (TAC) in brain tissue were determined as previously described (Kambayashi et al., 2009). Briefly, 90 μL of phosphate-buffered saline (10 mM, pH 7.2), 50 μL of myoglobin solution (18 μM), 20 μL of ABTS solution (3 mM), and 20 μL of standard or diluted homogenate were methodically added to a 96-well plate and mixed well at 25 1C for 3 min. Then, 20 μL of hydrogen peroxide was added to each well and incubated for 5 min. The absorbance was measured at 600 nm using a spectrophotometer (Molecular Devices). Levels of TAC were expressed as gallic acid equivalent antioxidant capacity (GEAC).

2.13. Determination of total GSH content, GSH-Rd, and GST in brain tissue GSH content in brain tissue was determined as previously described (Ellman, 1959). Briefly, 80 mL of NADPH (0.3 mM)/DTNB (4 mM) mixture (7:1) was combined with 50 mL of homogenate or GSH standard in a 96-well plate. Then, 20 mL (0.06 units) of GSHRd solution was added to each well. Absorbance was measured at 405 nm using UV spectrophotometer (Molecular Devices). GSH-Rd activity in brain tissue was determined as previously described (Smith et al., 1988). Briefly, 150 mL of GSSG (2 mM) and 30 mL of GSH-Rd assay buffer (100 mM potassium phosphate buffer, pH 7.5; containing 1 mM EDTA) were added to 30 mL of brain tissue homogenate and diluted with GSH-Rd dilution buffer (100 mM potassium phosphate buffer, pH 7.5; containing 1 mM EDTA and 1 mg/mL bovine serum albumin). After addition of 75 mL DTNB (3 mM) and 15 mL NADPH (2 mM), the absorbance measured at 412 nm using UV spectrophotometer (Molecular Devices). Enzyme activity was calculated using the following formula: enzyme activity (unit/mL) ¼ [(Δa sample Δa blank) (dilution factor)]/[14.15 mM 1/cm (volume of sample in mL)]. GST activity in brain tissue was measured using commercially available assay kit (GST Assay Kit; Sigma, Saint Louis, MI, USA) according to the manufacturer's protocol. Briefly, 196 μL of substrate solution (980 μL of DPBS, 10 μL of 200 mM reduced L-glutathione, and 10 μL of 100 mM CDNB) was mixed with 4 μL of brain tissue homogenate or standard (GST solutions of 0–250 μg/mL) in a 96-well plate. Absorbance was measured at 340 nm using UV spectrophotometer (Molecular Device Corp). Enzyme activity in the sample was calculated using the following formula: Enzyme activity: (unit/mL) ¼ [(ΔA340)/min 0.2 dilution factor]/(5.3 mM 1/cm volume of enzyme sample tested). 2.14. Determination of SOD and catalase in brain tissue SOD activity in brain tissue was determined by SOD assay kit (Dojindo Laboratories; Kumamoto, Japan) according to the manufacturer's protocol. Briefly, 20 μL of brain tissue was mixed with 200 μL of WST-1 working solution in 96-well plate. Subsequently, 20 μL of enzyme working solution was added and mixed well. The plate was incubated at 37 1C for 20 min, and the absorbance was measured at 450 nm using UV spectrophotometer (Molecular Devices). Dilutions of bovine erythrocyte SOD (Sigma) ranging (0.01–50 unit/mL) was used as standards. Catalase activity in brain tissue was determined as previously described (Wheeler et al., 1990). Briefly, 50 μL of phosphate buffer (250 mM, pH 7.0), 50 μL of methanol (12 mM), and 10 μL of H2O2 (44 mM) were mixed with 100 μL of sample or standard solutions in 1.5 mL tube for 10–20 min and was reaction was stopped by adding 150 μL of Purpald solution (22.8 mM Purpald in 2 N potassium hydroxide) incubated for 20 min at 25 1C, 150 μL of potassium periodate (65.2 mM in 0.5 N potassium hydrate) was added to the mixture. Absorbance of the purple formaldehyde product was measured using UV spectrophotometer at 550 nm (Molecular Devices). 2.15. Gene expression analysis by quantitative real-time PCR The expression of genes encoding mAChR subtypes 1–5 and inducible nitric oxide synthase (iNOS) was measured using quantitative real-time PCR. Total RNA was later isolated from brain tissue in RNA using an RNeasy Midi Kit (QIAGEN; Valencia, CA, USA). The cDNA was synthesized using a High-Capacity cDNA reverse transcription kit (Ambion; Austin, TX, USA). The real-time PCR was performed using SYBRGreen PCR Master Mix (Applied Biosystems; Foster City, CA, USA) and PCR amplification was



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Table 2 Sequence of the primers used in real-time PCR analysis. Gene (number)

Primer sequencing (forward and reverse)

Product size (base pair)

Annealing temperature (1C)

mAChR 1 (NM_001112697) mAChR 2 (NM_203491) mAChR 3 (NM_033269) mAChR 4 (NM_007699) mAChR 5 (NM_205783) iNOS (NM_010927) β-actin (NM_007393)

50 -AGT GGC ATT CAT CGG GAT CA-30 50 -CTT GAG CTC TGT GTT GAC CTT GA-30 50 -GCT GCG TGG GTT CTT TCC T-30 50 -GAA TGT AGC ACT CCC CGT CTT C-30 50 -GAG CCG GTG TGA TGA TTG GT-30 50 -GGC ACA GTT CTC TTC CCT ACA AA-30 50 -GGC CAA GAC TCT GGC TTT CC-30 50 -CTT CCC GTT GCG CAG TTC-30 50 -ATC ATT GGC ATC TTC TCC ATG A-30 50 -AGT CGA GTG CAA GCC AAA GG-30 50 -GGC AGC CTG TGA GAC CTT TG-30 50 -TGC ATT GGA AGT GAA GCG TTT-30 50 -GGC ACC ACA CCT TCT ACA ATG A-30 50 -ATC TTT TCA CGG TTG GCC TTA G-30

100

60

100

60

100

59

100

60

100

60

120

60

100

59

mAChR; muscarinic acetylcholine receptor, iNOS; inducible nitric oxide synthase, β-actin as a housekeeping gene.

performed using a standard protocol with the IQ5 PCR Thermal Cycler (Bio-Rad; Hercules, CA, USA). Primer sequences, product sizes, and annealing temperatures are summarized in Table 2.

2.16. Primary microglial cell culture Primary cerebral microglial cells were purified from neonatal rats according to standard procedure. Briefly, a 1-day-old postnatal Sprague–Dawley rat pup (Koatech; Gyeonggido, Korea) was decapitated in an ice-chilled dish. After removing the meninges, the brain was minced and dissociated by pipetting in Hank's balanced salt solution (HBSS; HBSS with 55.5 mM glucose, 20.4 mM sucrose, and 4.2 mM sodium bicarbonate). The collected cells were re-suspended in minimum essential medium (MEM; MEM with 100 μM non-essential amino acid solution, 2 mM L-glutamine, and antibiotics; containing 20% FBS) and cultured on poly-L-lysine-coated T75 flasks. After 7 days, the flasks were agitated on an orbital shaker for 2 h at 200 rpm at 37 1C, and the supernatant with floating cells was collected. After seeding, cells were incubated for 1 h, which allows the microglia to attach the dish. The non-adherent cells were removed by washing (astrocytes). Microglial cells were counted and seeded in MEM growth media (MEM with 100 μM non-essential amino acid solution, 1.4 mM L-glutamine, and antibiotics; containing 10% FBS). Under these conditions, the purity of the microglial population was 95% by immunofluorescence analysis. During the process, many antibodies and proteins were used to detect specific type of cells: anti-Iba1 for microglial cells, Anti-20 ,30 -cyclic nucleotide 30 -phospho-diesterase for oligodendrocytes, and glial fibrillary acidic protein for astrocytes.

2.17. Determination of ROS and NO production in microglial cells Microglial cells were plated at 1 106 cells/mL and treated with or without Myelophil (25, 50, 100, 200 μg/mL) for 2 h by incubating with 1 μg/mL LPS for 22 h. ROS production was measured using a dichlorodihydrofluorecein diacetate (DCFH2-DA) assay. After the removal of media from wells, the cells were incubated with 10 μM DCFH2-DA for 45 min. Fluorescence was measured (Wallac 1420, Perkin-Elmer; Turku, Finland) at an excitation wavelength of 485 nm and emission wavelength of 530 nm using spectrofluorometer. NO production was measured using Griess reagents. Absorbance was measured at 540 nm using UV spectrophotometer (Molecular Devices).

2.18. Statistical analyses All data's obtained were expressed as means 7 standard deviations (SDs). Statistically significant differences between the groups were analyzed by one-way analysis of variance (ANOVA) followed by post hoc multiple comparisons with Tukey's t-test using the IBM SPSS statistics 20.0 (SPSS Inc.; Chicago, IL, USA). Differences at Po 0.05, P o0.01, or P o0.001 were considered statistically significant.

3. Results 3.1. Morris water maze task The escape latency and path length of the group receiving scopolamine was significantly longer (approximately 2-fold in both) than the naive group in the fourth day (P o0.05 and Po 0.001, respectively). Scopolamine receiving group spent significantly less time in the target quadrant (by approximately 30%) compared with naive group (P o0.01). Pretreatment with Myelophil significantly ameliorated not only the delayed escape-latency time (Po 0.05 for both 100 and 200 mg/kg) but also increased the cumulative path-length (P o0.001 for both 100 and 200 mg/kg; Fig. 2A and B). In fifth day, pretreatment with Myelophil ameliorated the time spent in the target quadrant (P o0.01 for both 100 and 200 mg/kg; Fig. 2C). Tacrine had only similar effect on time spent in target quadrant. 3.2. Effect on AChE activity in brain tissue AChE activity in brain tissue was significantly increased (by approximately 1.5-fold) in the group receiving scopolamine compared with the naive group (P o0.001). Pretreatment with Myelophil completely modulated the increase of AChE activity in experimental compared with the control group (Po 0.01 for both 100 and 200 mg/kg). Tacrine also showed significant modulation of these alterations in brain tissue (Fig. 3A). 3.3. Western blot analysis of brain tissue The scopolamine treatment significantly suppressed the phosphorylation of ERK in brain tissue compared with naive group (P o0.001). The pretreatment with Myelophil significantly reversed the suppression of phosphorylated ERK compared with the control group (P o0.01 for both 100 and 200 mg/kg). Tacrine


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Fig. 2. Morris water maze task. (A) Escape latency of acquisition trial over 4 days. (B) Time spent in target quadrant of probe trial. Data are expressed as the means 7 SDs (n¼8). #Po 0.05, ##Po 0.01 compared with the naïve group; nP o 0.05, nnPo 0.01 compared with the control group.

Fig. 3. AChE activity and ERK signaling. (A) AChE activity in brain tissue. (B) Phosphorylation of ERK in brain tissue as determined by Western blot. Data are expressed as the means 7 SDs (n¼ 8). ###Po 0.001, compared with the naïve group; nnP o0.01, nnnP o 0.001 compared with the control group.



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also showed attenuation of these alterations in brain tissue (Fig. 3B). 3.4. Effects on ROS in serum and brain tissue and NO levels in brain tissue ROS levels in serum and brain tissue were significantly increased (2-folds and 1.5-folds approx, respectively) in the scopolamine receiving group compared with the naive group (Po0.001 and Po0.05, respectively). Pretreatment with Myelophil significantly attenuated the increase of serum ROS levels compared to control group (Po0.001 for both 100 and 200 mg/kg). Additionally, the increase in brain ROS levels was significantly attenuated following pretreatment with Myelophil (Po0.001 for both 100 and 200 mg/kg; Fig. 4A). Tacrine also resulted in significant attenuation of these alterations in serum and brain tissue. NO levels in brain tissue significantly increased (by approximately 1.7-fold) in the scopolamine compared with the naive group (Po0.001). The increase of NO levels in brain tissue was significantly ameliorated following pretreatment with Myelophil (Po0.05 and Po0.01 for 100 and 200 mg/kg, respectively; Fig. 4B). Tacrine also resulted in significant attenuation of these alterations in brain tissue. 3.5. Effects on MDA level and protein carbonyl contents in brain tissue MDA levels in brain tissue significantly increased (by approximately 2.5-fold) in the scopolamine compared with the naive

7

group (P o0.001). The increase in MDA levels was significantly attenuated in the group pretreated with Myelophil compared with control group (P o0.001 for both 100 and 200 mg/kg; Table 3). Tacrine also resulted in a significant attenuation of these alterations in brain tissue. The protein carbonyl contents in brain tissue were significantly increased (by approximately 1.6-fold) in the scopolamine compared with the naive group (P o0.01). Myelophil pretreated groups showed significant attenuation in protein carbonyl contents compared with the control group (Po 0.05 for 100 mg/kg; Table 3). Tacrine did not result in any significant changes in protein carbonyl contents. 3.6. Effects on biomarker profiles of antioxidants in brain tissue The scopolamine group showed a significant decrease in the TAC capacity of brain tissue compared with naive group (Po 0.05), whereas Myelophil pretreatment groups significantly attenuated the depletion in the experimental compared with the control group (P o0.05 for both 100 and 200 mg/kg). The scopolamine treatment also slightly decreased the GSH content, GST, GSH-Rd, catalase, and SOD activities compared with naive group. Pretreatment with Myelophil significantly or completely ameliorated these decreases compared with the control group (P o0.05 or P o0.01 or Po 0.001 for 50, 100, and 200 mg/kg; no effect for 50 and 200 mg/ kg of catalase). In particular, pretreatment with Myelophil resulted in higher GSH content and increased activities of GST, GSH-Rd, and catalase compared with the naive group. Tacrine also resulted in

Fig. 4. ROS and NO levels. (A) Serum and brain tissue ROS levels. (B) Brain tissue NO level. (C) ROS and (D) NO production in primary microglial cells. Data are expressed as the means7 SDs (n¼ 8). #P o0.05, ###Po 0.001 compared with the naïve group; nP o0.05, nnPo 0.01, nnnPo 0.001 compared with the control group.



8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

Table 3 Effects on biomarkers of oxidative stress and antioxidant in brain tissue. Treatment

Naive

Control

Myelophil (mg/kg) 50

MDA (μM/mg protein) Protein carbonyl contents (μM/mg protein) TAC (μM/mg protein) GSH (μM/mg protein) GSH-Rd (U/mg protein) GST (U/mg protein) SOD (U/mg protein) Catalase (U/mg protein)

###

Tacrine (mg/kg) 100

16.53 7 3.68

40.34 7 5.79

1.767 0.63

2.87 7 0.27

61.077 9.48

40.78 7 11.97

21.25 7 3.30

17.247 3.07

4.707 0.60

3.737 0.70

##

5.157 0.65

nnn

5.777 1.14

0.517 0.07

0.45 7 0.03

#

0.577 0.02

nnn

18.577 1.81

17.81 7 2.53

23.577 2.20

nnn

709.077 128.41

541.50 7 63.51

##

##

#

36.157 5.33 2.36 7 0.43

n

54.517 12.90 23.127 2.57

#

22.157 3.07 2.077 0.61 n

nn

615.22 7 173.57

200 nnn

nn

25.44 74.93 2.23 70.34

nnn

nn

23.747 5.79

nnn

2.54 7 0.62

nn

56.417 9.94

n

28.26 72.75

nnn

28.447 3.00

nnn

nn

5.98 71.21

nnn

5.667 0.60

nnn

0.647 0.12

nn

0.59 70.04

nnn

0.65 7 0.08

nnn

25.38 7 5.69

nn

24.05 73.91

62.007 13.51 27.42 7 3.66

nn

10

nnn

792.53 7 204.32

nn

61.55 711.98

nn

747.25 7129.90

22.067 2.20 nn

nn

730.30 7 101.01

nnn

Data are expressed as the mean 7 SD (n ¼8). MDA, malondialdehyde; TAC, total antioxidant capacity; GSH, glutathione; GSH-Rd, glutathione reductase; GST, glutathione S-transferase; SOD, superoxide dismutase. #

Po 0.05. P o0.01. Po 0.001 compared with the naïve group. n Po 0.05. nn Po 0.01. nnn Po 0.001 compared with the control group. ##

###

Fig. 5. Gene expression levels in brain tissue. Changes in the expression of mAChR subtypes 1–5 and iNOS measured by real time-PCR. The mRNA levels were normalized to that of β-actin. Data are expressed as the means 7SDs (n¼ 5). ##Po 0.01 compared with the naïve group; nPo 0.05, nnPo 0.01 compared with the control group. MYP, Myelophil; THA, tacrine.

a significant attenuation of these alterations (exception of catalase and SOD activities) in brain tissue (Table 3). 3.7. Effect on mRNA expression in brain tissue The expression of mAChR-1 in brain tissue was slightly downregulated in the scopolamine compared with the naive group. Pretreatment with Myelophil significantly modulated the alteration of mAChR-1 in the experimental compared with the control group (P o0.01 for 50, 100, and 200 mg/kg), but the expression levels of mAChR subtypes 2–5 were not affected. The expression of iNOS in brain tissue was significantly up-regulated (by approximately 1.6-fold) in the scopolamine group compared with the naive group (P o0.01). The expression of iNOS was notably decreased by pretreatment with Myelophil compared with control group (P o0.05 or P o0.01 for 50, 100, and 200 mg/kg; Fig. 5). 3.8. Effect on ROS and NO levels in primary microglial cells Myelophil did not exhibit cellular toxicity against microglial cells during the 24-h incubation (data not shown). ROS production

in primary microglial cells was significantly increased (2.5-folds approx) by lipopolysaccharide (LPS) treatment. Myelophil pretreatment group significantly attenuated ROS production compared with LPS-treated group (P o0.05 for 200 μg/mL; Fig. 4C). NO production in primary microglial cells was significantly increased (3.5-folds approx) in LPS-treated group compared with naive group (P o0.001). Myelophil pretreated groups significantly attenuated the NO production compared with the LPS-treated group (Po 0.05 for both 100 and 200 μg/mL; Fig. 4D). A graphical description of possible mechanisms of action involved in the Myelophil pharmacological activities are described in Fig. 6.

4. Discussion Neurodegenerative disorders are often characterized by the presence of both motor disturbances and memory defects. These symptoms are closely associated with dysfunction in the cholinergic system, including alterations in neurons, neurotransmitters and their receptors (Kihara and Shimohama, 2004; Shen, 2010). Treatment of neurodegenerative disorders, such as Alzheimer's



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Q2 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

Fig. 6. Summary of the pharmacological action of Myelophil.

disease, has focused on substances capable of inhibiting AChE activity, though these drugs often come with serious adverse effects including hepatotoxicity, diarrhea, insomnia, and vomiting (Ellis, 2005). Therefore a number of groups have begun investigating the therapeutic potential of herbal substances for the treatment of neurodegenerative disorders. Many of these herbs possess potent antioxidative properties, suggesting that these treatments may be useful for combating neurodegenerative disorders caused by oxidative stress (El-Sherbiny et al., 2003; Giridharan et al., 2011; Nunomura et al., 2006). As brain regions, such as the basal forebrain, hippocampus, and amygdala, are particularly vulnerable to oxidative stress (Mattson et al., 1999), therapeutics strategies capable of regulating oxidative stress may hold promise for patients with cognitive disorders. We investigated the protective effects of Myelophil on brain dysfunction and oxidative stress-associated injury following scopolamine injection (1 mg/kg, ip) in a mouse model of memory impairment. Scopolamine is an acetylcholine (ACh) inhibitor known to block the signals underlying memory (Blokland, 2005). To address the effects of Myelophil on memory and spatial learning, a Morris water maze was used (Morris, 1984). Escape latency was gradually prolonged following scopolamine injection; with escape time delayed two fold by day 4. This result was consistent with time spent in the target quadrant, showing a 30% decrease compared to naïve group. As expected, pretreatment with Myelophil not only ameliorated the decrease in escape latency time, but also increased time spent in the target quadrant. These results suggest that Myelophil may be useful for the prevention of memory defects. Previous studies have shown that scopolamine-induced memory impairment is an appropriate model for the study of the cholinergic dysfunction in both neurotransmitters and their receptors (Falsafi et al., 2012; Tota et al., 2012). ACh levels in the central cholinergic synapses need to be maintained to normalize memory function; however, the disruption of ACh by the overexpression of AChE can disrupt cholinergic activity in the synapse (Abreu-Villaca et al., 2011). In this study, AChE activity in brain tissue was notably increased by scopolamine; pretreatment with Myelophil led to modest attenuation of AChE activity in the brain. As AChE inhibitors have shown potential in rodent models of amnesia (Shi et al., 2010), the attenuation of AChE activity suggests that

9

Myelophil may confer anti-amnesic effects on scopolamineinduced memory impairment. ACh signaling occurs following binding of ACh to mAChRs in the synaptic cleft; the neuronal signal is then sequentially transferred to G-protein coupled receptors and ERK signaling pathways (Giovannini, 2006; Wess, 1993). ERK signaling is involved in a wide range of cellular functions, including neuronal proliferation, cell survival and death, neuronal synaptic plasticity, and memory (Impey et al., 1999). Activation of ERK in the hippocampus is suppressed by scopolamine (Moosavi et al., 2012); similar effects were seen our experiment. Pretreatment with Myelophil significantly ameliorated this suppression of ERK activities, as evidenced by an increase in the proportion of phosphorylated ERK protein. As ERK plays a significant role in the coordination of mAChR activity (Rosenblum et al., 2000), we also examined mAChR expression. Previous studies have revealed that mAChR-1knockout mice exhibit clear defects in working memory (Anagnostaras et al., 2003). In a clinical study, the genetic expression of mAChR-1 was decreased significantly in the temporal cortex of Alzheimer patients, whereas that of mAChR subtypes 2–4 was not changed (Wang et al., 1992). Here, expression of mAChR-1 was significantly decreased following scopolamine treatment; pretreatment with Myelophil restored mAChR expression in the brains of mice, consistent with its effects on other steps in this pathway. These findings suggest that Myelophil influences both the ERK signaling pathway and mAChR-mediated neuronal plasticity. Many factors affect the pathology of neurodegeneration, including immune activation, metabolism, vascular conditions, inflammation, and oxidative stress (Brown et al., 2005). Inflammatory responses play an important role in the degenerating CNS conditions such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (Kaushik and Basu, 2013). The microglia is key cellular mediators of the neuroinflammatory response, with both astrocytes and oligodendrocytes also playing an important role in neuroinflammation. The activation of microglia and astrocytes provokes NO production through up-regulation of iNOS (Saha and Pahan, 2006). This induction of neuroinflammation leads to substantial increases in oxidative stress, an important mediator of pathology in neurodegenerative disorders (Verri et al. 2012; Wang et al., 2013). Previous reports have revealed that immunomodulators could potentially contribute to the treatment of neurodegenerative diseases (Becker et al., 2013; Clark and Vissel, 2013). In the current study, markers of oxidative stress were significantly increased in brain tissue and sera following scopolamine treatment. ROS levels were significantly increased in the sera and brains of affected mice, and NO levels also increased in brain tissue. Additionally, scopolamine treatment significantly increased the production of MDA and protein carbonyl content in brain tissue. It is well known that end products of lipid peroxidation, MDA, and protein carbonyl content are commonly elevated in patients with neurodegenerative diseases (Gustaw-Rothenberg et al., 2010). Pretreatment with Myelophil significantly attenuated these abnormalities as evidenced by reduced levels of ROS, NO, MDA, and protein carbonyl content in the sera and/or brains of mice. Interestingly, the group pretreated with Myelophil showed lower levels of ROS in the brain than the naïve group. Similar results were also seen in in vitro studies examining primary microglia cells treated with Myelophil. These results are consistent with the down-regulation of iNOS gene expression seen in the brain following Myelophil treatment. Most biological organisms are well equipped with antioxidant systems that protect against oxidative stress-induced damage. Brain is particularly vulnerable to oxidative stress due to its high oxygen consumption, abundance of unsaturated fatty acids, and


10

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high concentrations of metal ions, while maintaining a relatively low antioxidant capacity (Choi, 1993; Halliwell, 1992; Hill and Switzer, 1984). In this study, scopolamine treatment significantly increased oxidative stress in the brains of affected mice as evidenced by decreases in TAC, total GSH contents, SOD, catalase, GSH-Rd, and GST activities. The mechanisms underlying the induction of oxidative stress following scopolamine treatment are not yet elucidated; however, many experiments have demonstrated the presence of oxidative stress in scopolamine-induced animal models (Giridharan et al., 2011; Harrison et al., 2009). In our study, altered biomarkers for oxidative stress were significantly restored by pretreatment with Myelophil. These results were similar to a previous report that Myelophil protects brain tissue against chronic restraint stressinduced oxidative brain injury (Lee et al., 2012), and against acute stress-induced oxidative liver injury (Kim et al., 2012). These results strongly support the antioxidant potential of Myelophil, which appears to be capable of reducing oxidative damage in the brain. Myelophil is composed of two medicinal plants, Astragali Radix and Salviae Miltiorrhizae Radix, which were traditionally thought to maintain the homeostasis of Qi and blood in the body. The Qi and blood are the two essential components of the human body according to the theory underpinning TCM (Cho et al., 2009). Clinically, Myelophil has been prescribed to treat fatigueassociated disorders, such as cancer or chronic fatigue syndrome. The primary clinical use of Myelophil is for treatment of chronic fatigue syndrome, a condition strongly associated with oxidative stress (Vecchiet et al., 2003). Previous studies have reported that Salviae Miltiorrhizae Radix and its main compound, salvianolic acid B, possess anti-amnesic and antioxidant effects via up-regulation of cholinergic receptors, and inhibition of free radical formation (Du et al., 2000; Kim et al., 2011). Additional research has confirmed that astragaloside IV, a major compound of Astragali Radix, is effective for preventing memory loss in a mouse model (Tohda et al., 2006). In this study, the pharmacological activity of Myelophil was evidenced in a mouse model of memory impairment.

5. Conclusion Despite the limitations of animal models, our results suggest that Myelophil exerts its anti-amnesic effects via maintenance of ACh levels, regulation of ERK-signaling pathways, and modulation of antioxidant activity in brain tissue. Further studies are necessary to identify the active compounds responsible for this activity, and to confirm these results in other models.

Acknowledgements This study was supported by a grant (B100045) from the Oriental Medicine R&D Project, Ministry of Health & Welfare, Republic of Korea. References Aarsland, D., Mosimann, U.P., McKeith, I.G., 2004. Role of cholinesterase inhibitors in Parkinson's disease and dementia with Lewy bodies. Journal of Geriatric Psychiatry and Neurology 17, 164–171. Abreu-Villaca, Y., Filgueiras, C.C., Manhaes, A.C., 2011. Developmental aspects of the cholinergic system. Behavioural Brain Research 221, 367–378. Anagnostaras, S.G., Murphy, G.G., Hamilton, S.E., Mitchell, S.L., Rahnama, N.P., Nathanson, N.M., Silva, A.J., 2003. Selective cognitive dysfunction in acetylcholine M1 muscarinic receptor mutant mice. Nature Neuroscience 6, 51–58. Bartus, R.T., Dean, R.L., Beer, B., Lippa, A.S., 1982. The cholinergic hypothesis of geriatric memory dysfunction. Science 217(408.17).

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