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ARTICLE IN PRESSG ModelEP-5272; No. of Pages 8

Journal of Ethnopharmacology xxx (2008) xxx–xxx

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

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D-501, a standardized extract of Scrophularia buergeriana has bothognitive-enhancing and antioxidant activities in mice given scopolamine

un Ju Jeonga, Choong Je Mab, Ki Yong Leea, Seung Hyun Kimc,ang Hyun Sunga, Young Choong Kima,∗

College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, San 56-1, Shillim-Dong, Gwanak-Gu, Seoul 151-742, Republic of KoreaSchool of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701, Republic of KoreaInstitute for Life Science, Elcom Science Co. Ltd., Seoul, Republic of Korea

r t i c l e i n f o

rticle history:eceived 19 May 2008eceived in revised form 5 September 2008ccepted 3 October 2008vailable online xxx

eywords:crophularia buergerianalzheimer’s disease

a b s t r a c t

Aim of the study: The cognitive-enhancing and antioxidant activities of KD-501, a standardized extract ofthe roots of Scrophularia buergeriana Miquel (Scrophulariceae) were investigated.Materials and methods: KD-501 was orally administered to amnesic mice induced by scopolamine and weperformed passive avoidance and the Morris water maze tests. To elucidate the mechanism of cognitive-enhancing activity, the effects of KD-501 on the activities of acetylcholinesterase and antioxidant enzymeswithin the cortex and hippocampus of mice were evaluated.Results: Acute and prolonged oral administration of KD-501 significantly ameliorated scopolamine-induced amnesia in passive avoidance test. In the Morris water maze test, acute and prolonged

ognitive-enhancing activitycetylcholinesterasentioxidant activityD-501

administration of KD-501 improved the impairment of spatial memory induced by scopolamine indicatedby the formation of reference and working memories. The activity of acetylcholinesterase was significantlyinhibited by KD-501 within the cortex and hippocampus. Moreover, the reduced activities or contents ofglutathione reductase, superoxide dismutase (SOD) and reduced GSH within the cortex and hippocampuscaused by scopolamine were elevated by the treatment of KD-501.Conclusions: Taken together, it could be postulated that KD-501 may exert its potent cognitive-enhancing

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. Introduction

Alzheimer’s disease, the most common type of degenerativeementia has been characterized by the progressive impairment ofognitive function and changes in behavior and social adaptabilityKatzman, 1986; Francis et al., 1999). The severity of cognitive dys-unction and memory loss in Alzheimer’s disease is associated withhe cholinergic hypofunction (Giovannini et al., 1997). Neuronaloss in the basal forebrain particularly within the septohippocampalcetylcholinergic systems involved in learning and memory pro-esses constitutes a pathological hallmark of Alzheimer’s disease.urrently, the treatment for Alzheimer’s disease is the administra-ion of acetylcholinesterase inhibitors that increase the availability

Please cite this article in press as: Jeong, E.J., et al., KD-501, a standardizedantioxidant activities in mice given scopolamine. J. Ethnopharmacol. (2008

f acetylcholine at cholinergic synapses. Such acetylcholinesterasenhibitors as physostigmine, tacrine and donepezil antagonize theegative effects of scopolamine on spatial memory in variousehavioral tests (Dawson et al., 1991; Braida et al., 1996; Bejar et

∗ Corresponding author. Tel.: +82 2 880 7842; fax: +82 2 888 2933.E-mail address: youngkim@snu.ac.kr (Y.C. Kim).

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378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2008.10.006

lcholinesterase and antioxidative actions.© 2008 Elsevier Ireland Ltd. All rights reserved.

l., 1999). Scopolamine, a muscarinic antagonist that induces cen-ral cholinergic blockade, produces a reversible and well-describedmpairment in both (i) maintaining attention and (ii) both process-ng of information and the acquisition of new knowledge in rodentsOhno and Watanabe, 1996; Bejar et al., 1999) and humans (Jones etl., 1991; Wesnes et al., 1991). The cognitive deterioration observedesembles the memory disturbances seen in Alzheimer’s disease.ence, scopolamine-induced amnesic mouse has been used as anxperimental model for Alzheimer’s disease (Beatty et al., 1986;bert and Kirch, 1998; Kopolman and Corn, 1998).

Recently, El-Sherbiny et al. (2003) reported that memory impair-ent in scopolamine-induced animal model is associated with

ltered status of brain oxidative stress. Strong evidence supportinghe involvement of oxidative stress in degenerative changes withinhe forebrain cholinergic system has been suggested (Mattson andedersen, 1998). The findings led to the notion that drugs with

extract of Scrophularia buergeriana has both cognitive-enhancing and), doi:10.1016/j.jep.2008.10.006

ntioxidative effects might be beneficial for preserving brain func-ion. Antioxidant enzymes such as superoxide dismutase (SOD),lutathione peroxidase and catalase as well as the low moleculareight reductant, alpha-tocopherol, glutathione (GSH) and ascor-ate are involved in the reduction of oxidative stress (Wilson, 1997).

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E.J. Jeong et al. / Journal of Ethn

ntioxidant enzymes display the reduced activities in affectedrain region of Alzheimer”s disease patients (Zemlan et al., 1989;appolla et al., 1992). Moreover, the reduction in the level ofntracellular oxidized protein under these conditions has beenssociated with the improvement of cognitive and/or psychomo-or functions (Carney et al., 1991). Thus, immense efforts have beenirected to find therapeutic agents that could reduce the oxida-ive damage and promote a functional recovery in degenerativeisorders.

In the course of searching natural products with memorynhancing activity using scopolamine-induced amnesic mouse asn experimental model for Alzheimer’s disease, it was found thathe ethanolic extract of the roots of Scrophularia buergeriana MiquelScrophulariceae) showed a significant memory enhancing activity.crophularia buergeriana is a strong-scented perennial or bienniallant native to northern China, Korea and Japan with an impor-ant role in the traditional medicine of several oriental cultures.he dried root of Scrophularia buergeriana has been used in ori-ntal medicine as a treatment for fever, swelling, constipation,haryngitis, neuritis and laryngitis (Duck and Ayensu, 1985; Jiansuew Medical College, 1997). Scrophularia buergeriana root producesoolness and supplies tonic for the yin and blood, especially, toe used in the case of kidney yin deficiency that is associatedith aging. Kidney yin deficiency is regarded as the origin of age-

elated memory loss in Chinese traditional medicine. We previouslyeported anti-amnesic activities of phenylpropanoids and iridoidlycosides isolated from Scrophularia buergeriana root on mem-ry deficit induced by scopolamine in mice (Kim et al., 2003a).oreover, these compounds showed significant neuroprotective

ctivities against neurotoxicity induced by glutamate in the pri-ary cultures of rat cortical cells (Kim and Kim, 2000; Kim et al.,

002, 2003a).On the basis of the previous reports, we have verified the

ognitive-enhancing activity of a standardized extract of Scrophu-aria buergeriana root (KD-501) by passive avoidance and the Morris

ater maze tests in the present study. To elucidate the mechanismf cognitive-enhancing activity, we assessed the effects of KD-501n acetylcholinesterase in the cortex and hippocampus of mice. Inddition, the effects of KD-501 on activities of glutathione perox-dase, glutathione reductase and SOD, and contents of brain GSH

ere evaluated followed by the Morris water maze test.

. Materials and methods

.1. Plant material and preparation of KD-501

The roots of Scrophularia buergeriana were purchased fromyongdong Oriental Herbal Market (Seoul, Korea) and identified by

ate Dr. Dae Suk Kim, Professor Emeritus, College of Pharmacy, Seoulational University. Voucher specimens (SNUPH-0303) document-

ng these collections have been deposited in the Herbarium of theedicinal Plant Garden, College of Pharmacy, Seoul National Uni-

ersity. The standardized extract of Scrophularia buergeriana rootas provided by Central Research Institute of Kwang Dong Pharma-

eutical (Korea). The powdered Scrophularia buergeriana root (10 g)as weighted accurately and extracted with 70% ethanol for 2 h at0 ◦C, using a reflux. The extract (2.4 g) was filtered and evaporated

n vacuum and then suspended in 0.5% carboxy methyl celluloseCMC) to the desired concentration just before use.

KD-501 was standardized on the basis of E-cinnamic acid (CA),

Please cite this article in press as: Jeong, E.J., et al., KD-501, a standardizedantioxidant activities in mice given scopolamine. J. Ethnopharmacol. (2008

-p-methoxycinnamic acid (MCA), E-harpagoside (Hs) and 8-O-E-p-methoxycinnamoyl)harpagide (Hg) which were reported as

ain constituents of Scrophularia buergeriana, using HPLC-dioderray detector–electrospray-MS (HPLC-DAD–ESI-MS) as previouslyeported (Lee et al., 2007).

Ooipt

PRESSmacology xxx (2008) xxx–xxx

.2. Sample treatment

ICR mice were randomly assigned to groups that received CMCnormal control group) or KD-501 once prior to testing for the acutedministration test, and received them for 15 days prior to testingor the prolonged administration test.

The treatment was performed until the first day of passive avoid-nce test and the last day of the Morris water maze test.

.3. Experimental animals

Male ICR (Harlan Sprague–Dawley; 4 weeks old) mice (Orient.o. Ltd., Gyunggi, Korea), weighing 25–30 g, were used after a 1-eek adaptation period (20–23 ◦C; 12 h light cycle from 09:00 to

1:00; food, Agribrand Purina Korea, and water ad libitum). Allxperiments (viz., passive avoidance test and the Morris wateraze test) and the method used for euthanasia were according to

he guidelines of the Committee on Care and Use of Laboratorynimals of the Seoul National University.

.4. Passive avoidance test

Training for and testing of passive avoidance performance werearried out in two identical light and dark square boxes (Gemini Saniego Inc., USA) as described in our previous report (Kim et al., 1999,003a, 2003b). The mice were initially placed in the light chambernd 10 s later the door between compartments was opened. Whenice entered the dark compartment, the door automatically closed

nd an electrical foot shock (0.1 mA/10 g body weight) for a timeeriod of 2 s was delivered through the stainless steel rods (onerial training). Ten mice were used per treatment. Mice receivedMC or KD-501 (3–200 mg/kg body weight, p.o.) 120 min beforehe training trial. After 90 min, amnesia was induced in mice withcopolamine (1 mg/kg body weight, dissolved in normal saline)iven subcutaneously. Twenty-four hours after the training trial,he mice were again placed in the light compartment. The escapeatency to enter the dark compartment was measured. If the miceid not enter the dark compartment within 180 s, the experimentas stopped.

.5. Morris water maze test

A spatial memory test was performed by the method of Morris1984) with minor modification described in our previous reportsKim et al., 1999, 2003b). The Morris water maze is a white circularool (100 cm in diameter and 45 cm in height) with a featureless

nner surface. The circular pool was filled with water in which00 ml of milk had been mixed to a height of 30 cm (20 ± 1 ◦C). Theool was divided into four quadrants of equal area. A white plat-orm (6 cm in diameter and 29 cm in height) was centered in onef the four quadrants of the pool and submerged 1 cm below theater surface so that it was invisible at water level. The continuous

ocation of each swimming mouse, from the start position to thelatform, was monitored by a video tracking system (Smart 2.5).

n the water maze experiments, the day prior to the experimentas dedicated to swim training for 60 s in the absence of the plat-

orm. In the days following, the mice were given two trial sessionsach day for 4 consecutive days. During each trial, the time taken towim to the platform (escape latency) was recorded. This param-ter was averaged for each session of trials and for each mouse.

extract of Scrophularia buergeriana has both cognitive-enhancing and), doi:10.1016/j.jep.2008.10.006

nce the mouse located the platform, it was permitted to remainn it for 10 s. If the mouse did not locate the platform within 120 s,t was placed on the platform for 10 s and then removed from theool by the experimenter (trial 1). The mouse was given secondrial (trial 2) with an inter-trial interval of 20 min for 4 consecutive

IN PRESSG ModelJ

opharmacology xxx (2008) xxx–xxx 3

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Fig. 1. The enhancing effects of acute and prolonged administration of KD-501 onmemory impairment induced by scopolamine in mice. 90 min before the train-ing trial, mice received KD-501 orally. After 60 min, amnesia was induced in micewith scopolamine (1 mg/kg body weight, s.c.). Twenty-four hours after the train-ing trial, the mice were again placed in the light compartment. The latency toenter the dark compartment was measured. The values shown are step throughlatency ± S.E.M. Results significantly differ from the values of scopolamine only-tK(s

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ARTICLEEP-5272; No. of Pages 8

E.J. Jeong et al. / Journal of Ethn

ays. The point of entry of the mouse into the pool and the locationf the platform for escape remained unchanged between trial 1 andrail 2 but was changed each day thereafter. The decrease in escapeatency from day to day in trial 1 represents long-term memoryr reference memory, while that from trial 1 to trial 2 representshort-term memory or working memory. Ten mice were used perreatment. Mice were treated with CMC or KD-501 (100 mg/kg bodyeight, p.o.) given before the training trial. After 90 min, amnesiaas induced in mice with scopolamine (1 mg/kg body weight, dis-

olved in normal saline) given subcutaneously. All mice were testedor spatial memory 30 min after the administration of scopolamine.

.6. Acetylcholinesterase activity determination

Mice were treated with CMC or KD-501 (100 mg/kg body weight,.o.). Ten mice were used per treatment. Acetylcholinesterase activ-

ty was measured by the method of Ellman et al. (1961) with slightodification (Cheng and Tang, 1998). The mice were euthanized

0 min after treatment and the brains were removed. The cerebralortex and hippocampus were dissected out from the brain. Botharts of the brain were rapidly homogenized, with sodium phos-hate buffer (0.1 mM, pH 7.4). Each homogenate was preincubated

or 5 min at 37 ◦C with 0.1 mM tetraisopropyl pyrophosphoramideTPPA), a selective inhibitor of butyrylcholinesterase. For the deter-

ination of acetylcholinesterase activity, a reaction mixture thatontained 470 �l sodium phosphate (0.1 mM, pH 8.0), 167 �lf 4% 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) and 33 �l ofomogenate was incubated at 37 ◦C for 5 min. Then, 280 �l of 1 mMcetylcholine iodide solution was added to the reaction mixture.fter the incubation for 3 min at 37 ◦C, the reaction was terminatedy adding 50 �l of 2 mM neostigmine. The acetylcholinesterasectivity was calculated as the optical density (OD) value per mgrotein (see above).

.7. Antioxidant assay

Following the Morris water maze test, mice were euthanized.he cerebral cortex and hippocampus were dissected rapidly undertandard conditions at 4 ◦C and homogenated in 0.1 M phosphateuffer (pH 7.4). The homogenates were centrifuged for 30 min at000 × g at 4 ◦C and the supernatant (cytosolic and mitochon-rial fractions) was collected for assessing antioxidative enzymectivity and GSH content. The activity of SOD was determinedccording to the method of McCord and Fridovich (1969) by theanthine–xanthine oxidase reaction. GSSG reductase activity waseasured according to the method of Carlberg and Mannervik

1975) based on the reduction of GSSG by GSSG reductase in theresence of NADPH. The activity of glutathione peroxidase wasetermined by quantifying the rate of oxidation of GSH to GSSG byumene hydroperoxide (Flohe and Gunzler, 1984). Values shownre the mean ± S.D. Protein concentrations were determined usingbicinchoninic acid (BCA) kit with bovine serum albumin as a

tandard (Smith et al., 1985). Total GSH in the supernatant wasetermined spectrophotometrically using the enzymatic cyclingethod (Tietz, 1969).

.8. Statistical analysis

Data for the Morris water maze and passive avoidance tests werexpressed as mean ± S.E.M. The activity of acetylcholinesterase and

Please cite this article in press as: Jeong, E.J., et al., KD-501, a standardizedantioxidant activities in mice given scopolamine. J. Ethnopharmacol. (2008

he levels of antioxidant values were expressed as mean ± S.D.assive avoidance latencies, acetylcholinesterase activity andntioxidant values were analyzed by one-way ANOVA. Morris wateraze latencies were analyzed by two-way ANOVA with the day as

ne variable and the treatment as a second. The data were consid-

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reated group: *P < 0.05, **P < 0.01 and ***P < 0.001. (A) Acute oral treatment ofD-501 (3–200 mg/kg body weight, p.o.) on scopolamine-induced amnesic mice.

B) Prolonged oral treatment of KD-501 (3–200 mg/kg body weight, p.o.) oncopolamine-induced amnesic mice.

red to be statistically significant if the probability had a value of.05 or less.

. Results

.1. Memory enhancing activity of KD-501 on memory deficit inice

The effect of KD-501 on the scopolamine-induced memoryeficit was evaluated using passive avoidance test. The step through

atency was shortened in scopolamine (1 mg/kg body weight s.c.)-reated mice compared to that of normal control mice (Fig. 1A). Thehortened step through latency induced by scopolamine was sig-ificantly restored by the acute treatment of KD-501 (3–200 mg/kgody weight, p.o.).

In mice treated with KD-501 at a dose of 30 mg/kg, the stephrough latency was increased to a level of 35.0% (89.9 ± 13.5) oformal control mice. The dosage increase over 30 mg/kg did notrolong the step through latency.

The prolonged treatment of mice with KD-501 showed the sig-ificant anti-amnesic activity at much lower doses compared tohat in the acute treatment (Fig. 1B). At a dose of 10 mg/kg, theecreased step through latency by scopolamine (1 mg/kg bodyeight) was increased to a level of 33.2% (71.4 ± 17.4) of normal

ontrol mice. At the highest effective dose of KD-501 (100 mg/kgody weight), the memory impairment was recovered up to 71.7%123.1 ± 21.6) of normal control mice.

.2. Anti-amnesic activity of KD-501 on impaired spatial memoryn mice

extract of Scrophularia buergeriana has both cognitive-enhancing and), doi:10.1016/j.jep.2008.10.006

On the basis of the results obtained from the passive avoidanceest, the efficacy of KD-501 in enhancing cognition after the impair-

ent of spatial memory via scopolamine was further evaluated.he mice were treated with KD-501 at the most effective dose inassive avoidance test, 100 mg/kg body weight, and performed the

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Fig. 2. The enhancing effect of acute administration of KD-501 (100 mg/kg body weight) on spatial memory impairment induced by scopolamine in mice. Mice were giventwo sessions of trials each day for 4 consecutive days. The swimming time required for mouse to escape to platform was recorded in each day. Each day, the mice were treatedw copola .E.M.( -501p g bodyi

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Table 1The effect of KD-501 on AChE activity within the cortex and hippocampus of mice.

Groups U/mg protein

Cortex Hippocampus

Control 0.6659 ± 0.0409 0.4808 ± 0.0211

ith KD-501 (100 mg/kg body weight, p.o.). After 90 min, amnesia was induced by sfter the injection of scopolamine. The values shown are the mean escape latency ± S10 ml/kg body weight, p.o.)–saline (1 mg/kg body weight, s.c.)-treated group. (B) KD.o.)–scopolamine (1 mg/kg body weight, s.c.)-treated group. (D) KD-501 (100 mg/k

njection.

orris water maze test. The escape latency for finding the plat-orm as described in Section 2 is depicted in Fig. 2. The escapeatencies of normal control mice or KD-501 only-treated mice wereapidly decreased after day 1. These two groups also exhibited aarked reduction in escape latencies from trial 1 and trial 2 on day

, and reached stable latencies after day 2 (Fig. 2A and B). By con-rast, the escape latency of mice treated with scopolamine (1 mg/kgody weight) did not significantly change during the 4 trainingays in which animals wasted the time exploring the margin of theool (Fig. 2C). Amnesic mice treated with KD-501 (100 mg/kg bodyeight, p.o.) demonstrated a significantly shortened interval to find

he platform as compared to mice given scopolamine only (Fig. 2D).he escape latencies were shortened in each daily first trial exhibit-ng the formation of reference memory. The reduction in escapeatencies from trial 1 and trial 2 was also observed on day 3.

In the acute treatment with KD-501, the effects on amnesic miceere significant with respect to treatment ([trial 1]: F(3, 144) = 42.9,< 0.0001; [trial 2]: F(3, 144) = 45.1, P < 0.0001), with respect toays ([trial 1]: F(3, 144) = 14.1, P < 0.0001; [trial 2]: F(3, 144) = 13.0,< 0.0001) and no effect with respect to the day and treatment

Please cite this article in press as: Jeong, E.J., et al., KD-501, a standardizedantioxidant activities in mice given scopolamine. J. Ethnopharmacol. (2008

nteraction ([trial 1]: F(9, 144) = 1.7, P < 0.2; [trial 2]: F(9, 144) = 1.3,< 0.3).

In the prolonged treatment experiment, daily oral treatmentf KD-501 (100 mg/kg body weight) to mice for 15 days producedo significant effect on the body weight compared to normal con-

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amine (1 mg/kg body weight, s.c.). All mice were tested for spatial memory 30 minResults significantly differ from the values in trial 1: *P < 0.05 and **P < 0.01. (A) CMC(100 mg/kg body weight, p.o.) alone treated group. (C) CMC (10 ml/kg body weight,weight, p.o.)-treated group 1 h before the scopolamine (1 mg/kg body weight, s.c.)

rol mice. Normal control mice and scopolamine-treated mice eachhowed very similar behavioral trends to those observed in thecute treatment experiment (Fig. 3A and C). The mice treated withD-501 alone showed similar pattern in escape latencies with nor-al control mice as well (Fig. 3B). The escape latency of mice treatedith KD-501 along with scopolamine was rapidly shortened afteray 1 and reached stable exhibiting well-formed reference mem-ry. The marked reduction in escape latencies from trial 1 and trialwas also observed on day 1 (Fig. 3D). Comparing with the result

extract of Scrophularia buergeriana has both cognitive-enhancing and), doi:10.1016/j.jep.2008.10.006

D-501 (100 mg/kg) 0.4550 ± 0.0154* 0.3273 ± 0.0067**

D-501 (200 mg/kg) 0.4339 ± 0.0120* 0.3261 ± 0.0125*

he values shown are the mean U/mg protein ± S.D.* Results significantly differ from the values of normal control group: P < 0.01.

** Results significantly differ from the values of normal control group: P < 0.001.

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Fig. 3. The enhancing effect of prolonged administration of KD-501 (100 mg/kg body weight) on spatial memory impairment induced by scopolamine in mice. Mice weregiven two sessions of trials each day for 4 consecutive days. The swimming time required for mouse to escape to platform was recorded in each day. Each day, the mice weretreated with KD-501 (100 mg/kg body weight, p.o.). After 90 min, amnesia was induced by scopolamine (1 mg/kg body weight, s.c.). All mice were tested for spatial memory3 ency ±( p. (B)w 100 mw

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0 min after the injection of scopolamine. The values shown are the mean escape latA) CMC (10 ml/kg body weight, p.o.)–saline (1 mg/kg body weight, s.c.)-treated groueight, p.o.)–scopolamine (1 mg/kg body weight, s.c.)-treated group. (D) KD-501 (eight, s.c.) injection.

In the prolonged treatment with KD-501, the effects onmnesic mice were significant with respect to treatment ([trial]: F(3, 144) = 39.9, P < 0.0001; [trial 2]: F(3, 144) = 35.1, P < 0.0001),ith respect to days ([trial 1]: F(3, 144) = 11.1, P < 0.0001; [trial 2]:

(3, 144) = 13.0, P < 0.0001) and no effect with respect to the daynd treatment interaction ([trial 1]: F(9, 144) = 1.7, P < 0.2; [trial 2]:(9, 144) = 1.3, P < 0.3).

.3. Acetylcholinesterase inhibitory effects of KD-501

Since acetylcholinesterase inhibitors are known to antagonizecopolamine-induced amnesia (Dawson et al., 1991; Braida et al.,996), the effect of KD-501 on acetylcholinesterase activities in theortex and hippocampus of mice was evaluated (Table 1). Acetyl-holinesterase activity within the cortex and hippocampus wasignificantly inhibited by 32% and 32%, respectively, by KD-501 at aose of 100 mg/kg and by 35% and 32% at a dose of 200 mg/kg whenompared to normal control. The statistics are shown in Table 1.

.4. Antioxidant effect of KD-501 on scopolamine-inducedxidative stress

To further elucidate the biochemical mechanism of the anti-

Please cite this article in press as: Jeong, E.J., et al., KD-501, a standardizedantioxidant activities in mice given scopolamine. J. Ethnopharmacol. (2008

mnesic activity of KD-501 in brain tissue, its effects on thectivities of antioxidant enzymes and on cellular GSH level wereetermined. The treatment of scopolamine resulted in a signifi-ant reduction of glutathione reductase and SOD activities in mouseortex and hippocampus, while the activity of glutathione perox-

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S.E.M. Results significantly differ from the values in trial 1: *P < 0.05 and **P < 0.01.KD-501 (100 mg/kg body weight, p.o.) alone treated group. (C) CMC (10 ml/kg bodyg/kg body weight, p.o.)-treated group 1 h before the scopolamine (1 mg/kg body

dase was virtually unchanged (Table 2). The acute treatment ofmnesic mice with KD-501 significantly preserved the activities oflutathione reductase and SOD decreased by scopolamine in theortex and hippocampus to a similar level of normal control mice.he reduction in GSH contents induced by scopolamine was alsoestored by the treatment with KD-501 (Table 3).

In the prolonged treatment with KD-501, the activities of glu-athione peroxidase, glutathione reductase and SOD were changedy the treatment of scopolamine showing very similar patterns tohose observed in acute treatment experiment as shown in Table 4.he prolonged treatment of amnesic mice with KD-501 signifi-antly recovered the reduced activities of glutathione reductasend SOD in cortex and hippocampus. The increase of SOD activ-ty was more remarkable in mice treated with KD-501 along withcopolamine than mice treated with KD-501 only. Unlike the acutereatment with KD-501 with/without scopolamine, the activity oflutathione peroxidase was increased in cortex and hippocampusTable 4). The reduced GSH contents induced by scopolamine werelso significantly reversed by the prolonged treatment with KD-501t much lower dose concentration (Table 5).

. Discussion and conclusions

extract of Scrophularia buergeriana has both cognitive-enhancing and), doi:10.1016/j.jep.2008.10.006

We previously reported a variety of neuroprotective compoundsuch as phenylpropanoids and iridoid glycosides isolated from Scro-hularia buergeriana root (Kim and Kim, 2000; Kim et al., 2002,003a). E-p-Methoxycinnamic acid and other phenylpropanoidsossessing an E-p-methoxycinnamoyl group in the molecule, sig-

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Table 2The effects of acute oral treatment of KD-501 on glutathione peroxidase, glutathione reductase and SOD activities within the cortex and hippocampus of scopolamine-inducedamnesic mice.

Groups Glutathione peroxidase (�mol NADPHoxidized/(min (mg protein)))

Glutathione reductase (�mol NADPHoxidized/(min (mg protein)))

SOD (�mol NADPH oxidized/(min (mg protein)))

Cortex Hippocampus Cortex Hippocampus Cortex Hippocampus

Control 0.738 ± 0.155 0.545 ± 0.056 14.577 ± 1.937 5.659 ± 0.571 10.785 ± 0.812 11.053 ± 0.742KD-501 0.617 ± 0.077 0.584 ± 0.052 13.460 ± 2.123 6.138 ± 1.103 11.781 ± 0.825 11.984 ± 1.079Sco 0.685 ± 0.087 0.549 ± 0.049 8.193 ± 0.754 3.989 ± 0.824 6.812 ± 0.896 6.683 ± 1.361Sco + KD-501 0.628 ± 0.107 0.516 ± 0.110 13.204 ± 1.446** 5.865 ± 0.767* 10.954 ± 0.931** 9.469 ± 0.806*

Each value represents the mean ± S.D. Sco: scopolamine.* Results significantly differ from the values of scopolamine only-treated group: P < 0.01.

** Results significantly differ from the values of scopolamine only-treated group: P < 0.001.

Table 3The effects of acute oral treatment of KD-501 on GSH level within the cortex and hippocampus of scopolamine-induced amnesic mice.

Groups Total GSH (nmol/(mg protein)) Reduced GSH (nmol/(mg protein)) GSSG/total GSH ratio

Cortex Hippocampus Cortex Hippocampus Cortex Hippocampus

Control 35.931 ± 5.925 11.481 ± 1.360 34.864 ± 5.765 10.901 ± 1.120 0.029 ± 0.006 0.048 ± 0.026KD-501 33.980 ± 2.943 10.808 ± 0.992 32.100 ± 3.063 10.271 ± 1.015 0.025 ± 0.010 0.050 ± 0.019Sco 25.532 ± 4.034 8.445 ± 0.992 23.334 ± 3.991 6.722 ± 1.341 0.087 ± 0.021 0.210 ± 0.074Sco + KD-501 32.923 ± 3.735 10.612 ± 0.951* 30.453 ± 3.825 10.062 ± 0.803** 0.075 ± 0.010 0.051 ± 0.025**

Each value represents the mean ± S.D. Sco: scopolamine.* Results significantly differ from the values of scopolamine only-treated group: P < 0.01.

** Results significantly differ from the values of scopolamine only-treated group: P < 0.001.

Table 4The effects of prolonged oral treatment of KD-501 on glutathione peroxidase, glutathione reductase and SOD activities within the cortex and hippocampus of scopolamine-induced amnesic mice.

Groups Glutathione peroxidase (�mol NADPHoxidized/(min (mg protein)))

Glutathione reductase (�mol NADPHoxidized/(min (mg protein)))

SOD (�mol NADPH oxidized/(min (mg protein)))

Cortex Hippocampus Cortex Hippocampus Cortex Hippocampus

Control 0.766 ± 0.010 0.420 ± 0.010 13.810 ± 1.028 9.326 ± 1.968 19.017 ± 1.015 7.426 ± 0.556KD-501 0.842 ± 0.038# 0.492 ± 0.014 15.522 ± 1.773# 8.211 ± 0.802 20.174 ± 1.464 10.510 ± 3.949##

Sco 0.758 ± 0.012 0.424 ± 0.013 6.955 ± 0.698 5.410 ± 1.057 17.018 ± 2.735 4.734 ± 1.069Sco + KD-501 0.826 ± 0.024* 0.406 ± 0.012 14.111 ± 1.455** 8.141 ± 0.973** 24.364 ± 2.442** 14.028 ± 2.499**

Each value represents the mean ± S.D. Sco: scopolamine.* < 0.0

< 0.0

npOprcoI

stt

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CKSS

E

Results significantly differ from the values of scopolamine only-treated group: P** Results significantly differ from the values of scopolamine only-treated group: P# Results significantly differ from the values of normal control group: P < 0.01.

## Results significantly differ from the values of normal control group: P < 0.001.

ificantly suppressed neurotoxicity induced by glutamate in therimary culture of rat cortical cells (Kim et al., 2002). Also, 8--(E-p-methoxycinnamoyl)harpagide and its aglycone, harpagiderotected cultured cortical cells from glutamate-induced neu-

Please cite this article in press as: Jeong, E.J., et al., KD-501, a standardizedantioxidant activities in mice given scopolamine. J. Ethnopharmacol. (2008

otoxicity (Kim et al., 2003b). Moreover, these neuroprotectiveompounds showed the significant anti-amnesic activity on mem-ry deficit induced by scopolamine in mice (Kim et al., 2003a).n this study, we evaluated the cognitive-enhancing activity of a

datt

able 5he effects of prolonged oral treatment of KD-501 on GSH level within the cortex and hip

roups Total GSH (nmol/(mg protein)) Reduced GSH

Cortex Hippocampus Cortex

ontrol 39.762 ± 2.912 8.946 ± 0.742 39.505 ± 2.9D-501 41.715 ± 2.023# 10.818 ± 1.015## 41.522 ± 2.0co 25.540 ± 2.722 6.621 ± 0.637 24.974 ± 2.7co + KD-501 36.964 ± 3.251** 7.863 ± 0.666* 36.785 ± 3.2

ach value represents the mean ± S.D. Sco: scopolamine.* Results significantly differ from the values of scopolamine only-treated group: P < 0.0

** Results significantly differ from the values of scopolamine only-treated group: P < 0.0# Results significantly differ from the values of normal control group: P < 0.01.

## Results significantly differ from the values of normal control group: P < 0.001.

1.01.

tandardized extract of Scrophularia buergeriana root (KD-501) con-aining these neuroprotective compounds by passive avoidance andhe Morris water maze tests.

The yield and antioxidative activity of natural extracts is closely

extract of Scrophularia buergeriana has both cognitive-enhancing and), doi:10.1016/j.jep.2008.10.006

ependent on the extract solvent (Julkunen-Tiito, 1985; Marinovand Yanishlieva, 1997; Muanza et al., 1998). This might be dueo the different polarity of the compounds with the activity ando the extraction of substances such as chlorophyll, dipeptides

pocampus of scopolamine-induced amnesic mice.

(nmol/(mg protein)) GSSG/total GSH ratio

Hippocampus Cortex Hippocampus

32 8.581 ± 0.694 0.006 ± 0.002 0.040 ± 0.00746# 10.265 ± 0.982## 0.004 ± 0.002# 0.051 ± 0.00903 5.253 ± 0.742 0.022 ± 0.004 0.209 ± 0.04850** 7.280 ± 0.564** 0.004 ± 0.001** 0.073 ± 0.015**

1.01.

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ARTICLEEP-5272; No. of Pages 8

E.J. Jeong et al. / Journal of Ethn

nd polyphenolics (Amarowicz and Shahidi, 1997; Kansci et al.,997; Hattori et al., 1998). The extract of Scrophularia buergerianaoot obtained with 70% ethanol showed the most potent anti-mnesic activity in passive avoidant test (data not shown). Hence,n this study, we evaluated the cognitive-enhancing activity of 70%thanolic extract of Scrophularia buergeriana root including theseeuroprotective and anti-amnesic constituents through passivevoidance and the Morris water maze tests. 70% ethanolic extract ofcrophularia buergeriana root was standardized and named KD-501.

Acute and prolonged treatment with KD-501 (3–200 mg/kgody weight) exhibited mitigation of memory deficit in mice

nduced by scopolamine in passive avoidance test. At the mostffective dose, the acute treatment with KD-501 increased thetep through latency to a level of 35% of normal control mice andhich was increased up to 71% at the prolonged treatment. KD-

01 also mitigated the impairment of spatial memory induced bycopolamine in mice as measured in the Morris water maze test.he acute and prolonged treatment of amnesic mice with KD-501100 mg/kg body weight, p.o.) significantly improved the deficits ineference and working memories. The prolonged treatment withD-501 shortened the escape latency from the first trial day, while

he reduction was significant on day 3 in acute treatment. Moreover,n passive avoidance test, the memory deficits induced by scopo-amine were reversed significantly by the prolonged treatment withD-501 at a dose of 3 mg/kg. Considering these results, it could beuggested that the prolonged treatment of KD-501 might slowlynduce adaptive cellular change within the central nervous system.t was also inferred that KD-501 may not have an ability to improver deteriorate learning and memory in normal control mice throughehavioral results of KD-501 only-treated mice in passive avoidancend the Morris water maze tests.

It has been demonstrated that impairments in learning, mem-ry and behavior observed in patients with dementia are caused, ateast in part, by changes within the cholinergic system (Blokland,995; Fodale et al., 2006). It has been demonstrated by previ-us animal and human studies that learning and memory cane modified by drugs affecting the central cholinergic systemBartus et al., 1982; Collerton, 1986). Cholinergic transmission iserminated mainly by acetylcholine hydrolysis via the enzymecetylcholinesterase. This enzyme is essential in maintaining theormal function of the nervous system, since it rapidly terminateshe action of acetylcholine released into the synapse. It is currentlyelieved that the action of this enzyme could affect the underlyingrocesses in Alzheimer’s disease (Ballard et al., 2005). Conse-uently, acetylcholinesterase has been a potential target for thereatment of Alzheimer’s disease and for prevention strategies. Thiss why we evaluated the effect of KD-501 on acetylcholinesterasectivity and correlated this activity with its anti-amnesic activities.D-501 inhibited acetylcholinesterase activity significantly in theippocampus by 32% as compared to normal control mice.

Many clinical studies have reported strong evidences that oxida-ive stress is involved in the pathogenesis of Alzheimer’s disease.ecently, El-Sherbiny et al. (2003) reported that memory impair-ent induced by acute administration of scopolamine in rats is

ssociated with altered level of GSH in the brain and with thectivities of antioxidant enzymes. Elevation of brain oxidative sta-us of amnesic rats resembled the clinical pathology observed inlzheimer’s disease patients (Palmer, 1999). In our experimentonditions, the treatment of scopolamine resulted in a signifi-ant decrease of both glutathione reductase and SOD activities,

Please cite this article in press as: Jeong, E.J., et al., KD-501, a standardizedantioxidant activities in mice given scopolamine. J. Ethnopharmacol. (2008

hile the activity of glutathione peroxidase was little changed.he administration of KD-501 significantly preserved the activityf glutathione reductase in the cortex and hippocampus to a sim-lar level observed in normal control mice. Although the reducedctivity of glutathione peroxidase induced by scopolamine was lit-

C

PRESSmacology xxx (2008) xxx–xxx 7

le changed by the acute treatment with KD-501, it was increasedy the prolonged treatment. The reduced contents of GSH inducedy scopolamine were also recovered by the treatment with KD-01. Glutathione peroxidase reduces toxic radicals using GSH as aubstrate, subsequent to the oxidation of GSH to GSSG. GSSG is inurn reduced again to GSH by glutathione reductase at the expensef NADPH, forming a redox cycle (Lu, 1999). We suggest that theestoration of activities of glutathione peroxidase and glutathioneeductase by KD-501 might promote scavenging of free radicalssing recycled GSH from GSSG. In accordance with this hypoth-sis regarding the effects of KD-501 on glutathione peroxidase andlutathione reductase activity, we showed that the depletion ofrain GSH which normally accompanies scopolamine treatmentas restored significantly by KD-501.

The most remarkable effect of KD-501 is the increased activity ofOD in cortex and hippocampus. Treatment with KD-501 not onlyreserved the reduced SOD activity induced by scopolamine butlso elevated it higher than that of normal control mice in the pro-onged treatment. In the brain of Alzheimer’s disease patients, SODctivity was significantly lower than that of non-demented controln the cerebellum (−27%), frontal cortex (−27%) and hippocam-us (−35%) (Chen et al., 1994). In this report, the marked changef SOD activity was found in the specific localized region withinhe brain of Alzheimer’s disease patients. Although there are stillonflicting reports associated with the SOD activity in Alzheimer’sisease, most recently, SOD mimetics have come to the forefrontf antioxidative therapeutics of neurodegenerative diseases (Pong,003). From the above results, it could be postulated that KD-01 scavenges ROS and exerts a protective effect against oxidativeamage induced by scopolamine by maintaining the activities oflutathione reductase and SOD. The cognitive-enhancing activitiesf KD-501 might result, in part, from the inhibition of acetyl-holinesterase activity, and the reduction in ROS by recovering thentioxidative defense system.

These results suggested that KD-501 might offer a usefulherapeutic choice in either the prevention or the treatment oflzheimer’s disease.

cknowledgement

This study was supported by a grant of the Korea Health 21 R&Droject, Ministry of Health & Welfare, Republic of Korea (A050599).

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