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Keywords:

  • cholinesterase inhibition;
  • Ecklonia stolonifera;
  • phlorotannins;
  • seaweeds;
  • sterols

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

ABSTRACT:  As part of this study on the isolation of cholinesterase inhibitors from natural marine products, the bioactivity of the ethanolic extracts from 27 Korean seaweeds were screened using acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory assays. Ecklonia stolonifera exhibited promising inhibitory properties against both AChE and BChE. Bioassay-guided fractionation of the active n-hexane and ethyl acetate (EtOAc) soluble fractions, obtained from the ethanolic extract of E. stolonifera, resulted in the isolation of the sterols; fucosterol (1) and 24-hydroperoxy 24-vinylcholesterol (2), from the n-hexane fraction and the phlorotannins; phloroglucinol (3), eckstolonol (4), eckol (5), phlorofucofuroeckol-A (6), dieckol (7), triphlorethol-A (8), 2-phloroeckol (9) and 7-phloroeckol (10), from the EtOAc fraction. Of these, compounds 2, 9 and 10 were isolated from E. stolonifera for the first time. Compounds 47, 9 and 10 exhibited inhibitory potential against AChE, with 50% inhibition concentration (IC50) values of 42.66 ± 8.48, 20.56 ± 5.61, 4.89 ± 2.28, 17.11 ± 3.24, 38.13 ± 4.95 and 21.11 ± 4.16 μM, respectively; whereas, compounds 1, 2, 4 and 6 were found to be active against BChE, with IC50 values of 421.72 ± 1.43, 176.46 ± 2.51, 230.27 ± 3.52 and 136.71 ± 3.33 μM, respectively. It has been suggested that the inhibition of these enzymes by the sterols and phlorotannins derived from marine brown algae could be a useful approach for the treatment of Alzheimer's disease.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

Alzheimer's disease (AD) is an irreversible, progressive neurodegenerative disorder, which occurs gradually, resulting in memory loss, behavior disturbances, personality changes and a decline in cognitive abilities; it is also the most common cause of dementia among the elderly.1 Neuropathological studies have demonstrated that cholinergic functions in the basal forebrain and cortex decline due to senile dementia during AD.2,3 The most promising therapeutic strategy for activating the cholinergic functions has been the use of cholinomimatic agents. For the treatment of AD, synthetic cholinesterase inhibitors, including tacrine, donepezil, rivastigmine and galantamine, are usually prescribed. However, the use of these drugs is limited due to their adverse side-effects, such as hepatotoxicity, gastrointestinal disturbance and problems with bioavailability.4–6 Due to the side-effects of synthetic cholinesterase (ChEs) inhibitors, and considering the preventive potential discovered in foods, herbs and seaweeds as bioactive sources, the isolation of better ChEs inhibitors from natural products still arouses great interest. Several classes of ChEs inhibitory compounds have been discovered, including alkaloids,7–13 farnesylacetone derivatives,14 pyrazoline derivatives,15 withanolides,16 terpenoids,17–22 shikimate derivates,23–26 flavonoids27 and sterols.28

Marine algae have been used for various purposes in food, industry and medicine.29–31 Recently, edible seaweeds have been shown to exert many positive physiological effects, including antidiabetic complication,32 antitumor,33 hepatoprotective,34 antiviral,35 antiplasmin inhibitory,36,37 algicidal,38 tyrosinase inhibitory,39 anti-inflammatory,40 nitrite-scavenging,41 antiskin aging,42,43 anticoagulative,44 antiproliferative,45 antioxidant46,47 and antiallergic activities.48

Ecklonia stolonifera Okamura is a perennial brown alga, belonging to the family Laminariaceae. This species is usually found in subtidal zones at depths of between 2 and 10 m, and is widely distributed throughout the eastern and southern coasts of Korea.49 It is frequently used as a foodstuff, along with Laminaria japonica and Undaria pinnatifida. Phloroglucinol, as well as phlorotannins, including eckstolonol, eckol, phlorofucofuroeckol A, dieckol and ecklonialactones, have all previously been isolated from E. stolonifera, and are responsible for many of the positive physiological effects associated with seaweeds.32,34,35,39,41–43,46–48,50–52

In the course of this study's search for ChEs inhibitors from natural marine products, the ethanolic extract of E. stolonifera was found to exhibit promising ChEs inhibitory activity. Further bioassay-directed purification of this extract, using a variety of chromatographic techniques, resulted in the isolation of two sterols and eight phlorotannins. Herein, the isolation and biological activities of these compounds are discussed.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

General experimental procedures

The 1H- and 13C-NMR spectra were measured using a JEOL JNM-EOP 400 Spectrometer (Tokyo, Japan) (400 MHz for 1H and 100 MHz for 13C). The chemical shifts were referenced to the respective residual solvent peaks, with the values recorded in δ. Column chromatography was carried out using silica gel 60 (70–230 mesh; Merck, Darmstadt, Germany), RP-18 Lichroprep (Merck), and Sephadex LH-20 (25–100 μ; Sigma, St. Louis, MO, USA). Thin-layer chromatography was carried out on precoated Merck Kieselgle 60 F254 plates (0.25 mm, Merck), with spots detected under UV light using 50% H2SO4 reagent. All solvents for the column chromatography were of regent grade, and acquired from commercial sources.

Materials

The leafy thalli of E. stolonifera were collected at Gijang-gun, Busan, during February 2000, and authenticated by Professor H. G. Kim of the Faculty of Marine Bioscience and Technology, Kangnung National University. All other leafy algal thalli were harvested at Chungsapo, Busan, during February 2003, and authenticated by an algologist, Professor C. H. Sohn, of the Fisheries Marine Life Sciences, Pukyong National University. Voucher specimens (No. 20000228) were deposited in the author's laboratory (Professor J. S. Choi).

Chemicals

Electric-eel AChE (EC 3. 1. 1. 7), horse-serum BChE (EC 3. 1. 1. 8), acetylthiocholine iodide (ACh), butyrylthiocholine chloride (BCh), 5,5′-dithiobis [2-nitrobenzoic acid] (DTNB), eserine, tacrine and berberine were purchased from Sigma (St. Louis, MO, USA). All other chemicals and solvents were high grade.

Extraction and isolation of compounds

After washing with sea and tap water, followed by drying in the shade, all of the seaweed samples for testing (100 g) were extracted with 95% ethanol. The weights of the ethanolic extracts are listed in Table 1.

Table 1.  Cholinesterase inhibitory activities of ethanolic extracts from selected Korean seaweeds at 100.0 μg/mL
PhylumSeaweedsYields (g)Inhibition (%) Mean ± SEM
AChEBChE
  • AChE, acetylcholinesterase; BChE, butyrylcholinesterase.

  • Yields of the ethanolic extracts of dried seaweed samples (100 g).

  • Mean ± standard mean error of three assays.

  • §

    Inhibition of the positive control; eserine showed 50% inhibitory activity against AChE at 0.0064 μg/mL, obtained by interpolation of the concentration-inhibition curve.

ChlorophytaCapsosiphon fulvescens (C. Agardh) Setchell & N.L. Gardner4.95−6.23 ± 1.6313.55 ± 4.25
Ulva pertusa Kjellman2.36−2.35 ± 1.49−16.99 ± 1.45
Enteromorpha linza (Linnaeus) J. Agardh3.50−14.12 ± 6.66−12.93 ± 1.11
Enteromorpha prolifera (Mueller) J. Agardh10.21−16.38 ± 4.15−10.91 ± 0.86
Codium fragile (Suringar) Hariot10.06−6.61 ± 6.80−25.23 ± 3.09
PhaeophytaUndaria pinnatifida (Harvey) Suringar2.09−1.80 ± 5.474.59 ± 0.27
Pelvetia siliquosa Tseng & Chang2.881.77 ± 5.3122.10 ± 0.29
Sargassum fulvellum (Turner) C. Agardh18.042.75 ± 2.53−10.80 ± 0.30
Sargassum horneri (Turner) C. Agardh20.27−16.12 ± 1.38−23.91 ± 0.78
Sargassum thunbergii (Mertens ex Roth) Kuntze1.668.38 ± 1.01−18.69 ± 2.12
Ishige okamurae Yendo2.26−21.94 ± 6.56−12.63 ± 1.50
Ecklonia cava Kjellman12.5021.28 ± 2.2113.14 ± 2.24
Ecklonia stolonifera Okamura8.9445.97 ± 3.1030.90 ± 4.20
Laminaria japonica Areschoug2.28−5.51 ± 6.994.02 ± 4.89
RhodophytaAhnfeltiopsis flabeliformis (Harvey) Masuda1.70−10.12 ± 0.625.46 ± 4.39
Callophyllis japonica Okamura2.46−5.91 ± 1.98−13.15 ± 1.37
Chondracanthus tenellus (Harvey) Hommersand5.10−18.90 ± 1.19−2.20 ± 3.21
Chondrus ocellatus Holmes5.897.19 ± 4.31−33.22 ± 2.00
Chondrus pinnulatus (Harvey) Okamura10.017.74 ± 4.25−21.46 ± 1.91
Meristotheca papulosa (Montagne) J. Agardh3.54−18.78 ± 0.71−17.60 ± 2.04
Gloiopeltis furcata (Postels & Ruprecht) J. Agardh5.34−6.09 ± 3.05−15.89 ± 2.28
Gelidium amansii (J.V. Lamouroux) J.V. Lamouroux0.199.94 ± 3.0923.55 ± 0.76
Gracilaria verrucosa (Hudson) Papenfuss8.378.35 ± 1.446.65 ± 4.35
Corallina officinalis Linnaeus1.374.23 ± 1.77−16.50 ± 1.28
Chondria crassicaulis Harvey13.29−3.13 ± 4.67−29.30 ± 4.17
Symphyocladia latiuscula (Harvey) Yamada3.17−2.81 ± 1.26−35.43 ± 1.29
Porphyra tenera Kjellman2.28−6.00 ± 3.9715.83 ± 2.89
Positive control§Eserine55.09 ± 1.1133.47 ± 5.55

For further chemical investigation, a lyophilized powder (3 kg) of E. stolonifera was extracted three times with 9 L of hot ethanol (3 × 9 L), followed by partitioning with organic solvents, yielding n-hexane (27.9 g), dichloromethane (CH2Cl2, 25.6 g), ethyl acetate (EtOAc, 25.0 g) and n-butanol (n-BuOH, 99.6 g) fractions. The n-hexane fraction, which exhibited profound BChE inhibitory activity, was subjected to chromatography on a silica gel column, with CH2Cl2 : methanol (20:1, 10:1, 10:3) as the eluent, yielding 13 subfractions (HF01–HF13). Subfractions HF03 and HF04 were combined, and then recrystallized with CH2Cl2 and methanol to yield fucosterol (1, 300 mg). Column chromatography of HF05 (1.64 g) on a silica gel column, with n-hexane : EtOAc (5:1) as eluent, resulted in the isolation of 24-hydroperoxy 24-vinylcholestesrol (2, 50 mg). The EtOAc fraction, which exhibited profound AChE inhibitory activity, was subjected to chromatography on a silica gel column, with EtOAc : methanol (50:1–5:1) as eluent, yielding 10 subfractions (EF01–EF10). Repeated column chromatography of EF01 (3.44 g) was conducted with a solvent mixture of n-hexane and EtOAc, yielding 11 subfractions (EF0101–EF0111). Phloroglucinol (3, 98 mg) was purified from EF0104 (250 mg) on an RP-18 column, eluted with aqueous methanol (20–100% methanol, gradient). RP-18 column chromatography of EF0105 (1.01 g), using identical solvent conditions, led to the isolation of eckstolonol (4, 60 mg), eckol (5, 135 mg) and 2-phloroeckol (9, 9 mg). Phlorofucofuroeckol A (6, 57 mg), dieckol (7, 87 mg) and 7-phloroecckol (10, 20 mg) were purified from EF0106 (945 mg), on RP-18 (20–100% methanol, gradient elution) and Sephadex LH-20 (100% methanol elution) columns. EF02 (634 mg) was subjected to chromatography on a silica gel column, with CH2Cl2 : methanol (18:1) as eluent, yielding eight subfractions (EF0201–EF0208). Successive chromatography of EF0204 (443 mg) on an RP-18 column, with 20% methanol as eluent, yielded five subfractions (F020401–F020405). Repeated column chromatography of EF020401 (111 mg) on a silica gel column, with CH2Cl2 : methanol (18:1) as eluent, resulted in the isolation of triphlorethol-A (8, 60 mg).

In vitro ChEs inhibitory activity assay

The inhibitory activities the ChEs were measured using the spectrophotometric method developed by Ellman et al.53 ACh and BCh were used as the substrates to assay the inhibitions of AChE and BChE, respectively. The reaction mixture contained: 140 μL of sodium phosphate buffer (pH 8.0), 20 μL of test sample solution and 20 μL of either AChE or BChE solution, which were mixed and incubated for 15 min at room temperature. The reactions were then initiated with the addition of 10 μL of DTNB, and 10 μL of either ACh or BCh, respectively. The hydrolysis of ACh or BCh was monitored by following the formation of the yellow 5-thio-2-nitrobenzoate anion at 412 nm for 15 min, which resulted from the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of either ACh or BCh, respectively. Test samples and the positive control (eserine, tacrine, berberine) were dissolved in 10% analytical grade ethanol. All reactions were performed in triplicate in 96-well microplates, using VERSA max (Molecular Devices, CA, USA). The percentage (%) inhibition was calculated from (E–S)/E × 100, where E and S are the enzyme activities without and with the test sample, respectively. The ChEs inhibitory activity of each sample was expressed in terms of the IC50 value (μg/mL or μM required to inhibit the hydrolysis of the substrate; ACh or BCh, by 50%), as calculated from the log-dose inhibition curve.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

For the potential isolation of ChEs inhibitors from natural marine products, the ethanolic extracts of 27 Korean seaweeds were evaluated using a bioassay-guided fractionation strategy. The ChEs inhibitory properties of the ethanolic extracts, observed at concentrations of 100.0 μg/mL, are listed in Table 1. Of the seaweeds tested, the ethanolic extracts of E. stolonifera and E. cava, belonging to the Ecklonia species, exhibited detectable ChEs inhibitory activities against both AChE and BChE, with inhibition of 45.97 ± 3.10 and 30.90 ± 4.20%, and 21.28 ± 2.21 and 13.14 ±  2.24%, respectively. In particular, E. stolonifera showed more potent ChEs inhibitory activity than E. cava, probably due to the compositional difference between their active components. Although Sargassum sagamianum has previously been reported to exhibit BChE inhibitory activity,14 our studies detected no such effects with the Sargassum sp. tested, i.e. S. fulvellum, S. horneri and S. thunbergii, which may be attributable to differences in the constituents between the species. Therefore, the ethanolic extract of E. stolonifera was chosen for further studies.

In the present ongoing study for the identification of active components, the ethanolic extract of E. stolonifera was successively partitioned with n-hexane, CH2Cl2, EtOAc and n-BuOH, yielding the respective fractions, with the inherent inhibitory activities of each then assessed. As shown in Table 2, the CH2Cl2 (71.52 ± 5.46 μg/mL), EtOAc (26.46 ± 4.76 μg/mL) and n-BuOH (82.15 ±  3.23 μg/mL) soluble fractions exhibited significant AChE inhibitory activities. The EtOAc soluble fraction of the ethanolic extract from E. stolonifera had noticeable inhibitory activity towards AChE. Conversely, the n-hexane (68.40 ± 4.03 μg/mL) and CH2Cl2 (71.10 ± 3.54 μg/mL) fractions had profound inhibitory activity against BChE. However, the H2O fraction showed no detectable activity towards the ChEs, suggesting the active ingredients of the EtOH extract of E. stolonifera were included in the moderately polar EtOAc fraction and non-polar n-hexane fraction for AChE and BChE, respectively. The comparative inhibitory activities toward ChEs of the fractions derived from the EtOH extract of E. stolonifera can be explained by the presence of different components. The activities of both the EtOAc and n-hexane fractions indicated that the inhibitory principles were phlorotannins and sterols, as they are known to widely exist in Ecklonia sp. and have polar solubilities in EtOAc and n-hexane, respectively.47,54 Therefore, the most active EtOAc and n-hexane soluble fractions were subjected to further chemical analysis, with repeated column chromatography of the active n-hexane and EtOAc fractions leading to the isolation of two sterols, fucosterol (1) and 24-hydroperoxy 24-vinylcholesterol (2), from the n-hexane fraction, and eight phlorotannins, phloroglucinol (3), eckstolonol (4), eckol (5), phlorofucofuroeckol-A (6), dieckol (7), triphlorethol-A (8), 2-phloroeckol (9) and 7-phloroeckol (10), from the EtOAc fraction. Of these compounds, 24-hydroperoxy 24-vinylcholesterol (2), 2-phloroeckol (9) and 7-phlorockol (10) were isolated from E. stolonifera for the first time in this study; their structures were also verified by comparison with published data.32,55,56 The structures of the isolated compounds are presented in Figure 1, with their ChEs inhibitory effects shown in Table 3. Of the isolated compounds, compounds 47, 9 and 10 exhibited significant AChE inhibitory activities, with IC50 values of 42.66 ± 8.48, 20.56 ± 5.61, 4.89 ± 2.28, 17.11 ± 3.24, 38.13 ± 4.95 and 21.11 ± 4.16 μM, respectively. In the AChE assay, compound 2 showed marginal inhibitory activity, with an IC50 value of 389.10 ± 2.29 μM. Conversely, compounds 1, 2, 4 and 6 exhibited moderate inhibitory activities against BChE, with IC50 values of 421.72 ± 1.43, 176.46 ± 2.51, 230.27 ± 3.52 and 136.71 ± 3.32 μM, respectively. However, compounds 3 and 8 showed no activity toward either AChE or BChE.

Table 2.  Cholinesterase inhibitory activities of the ethanolic extract and its fractions from Ecklonia stolonifera
FractionsIC50 (μg/mL) ± SEM
AChEBChE
  • AChE, acetylcholinesterase; BChE, butyrylcholinesterase.

  • Mean ± standard mean error of three assays.

  • Positive control of ChEs.

Ethanolic extract108.11 ± 2.35161.54 ± 4.20
n-Hexane fraction>30068.40 ± 4.03
CH2Cl2 fraction71.52 ± 5.4671.10 ± 3.54
EtOAc fraction26.49 ± 4.76173.50 ± 5.04
n-BuOH fraction82.15 ± 3.23258.80 ± 4.03
H2O fraction>300>300
Eserine0.004 ± 0.0010.02 ± 0.004
image

Figure 1. Structures of sterols and phlorotannins isolated from Ecklonia stolonifera.

Download figure to PowerPoint

Table 3.  Cholinesterase inhibitory activities of the sterols and phlorotannins isolated from Ecklonia stolonifera
CompoundsIC50 (μM) ± SEM
AChEBChE
  • AChE, acetylcholinesterase; BChE, butyrylcholinesterase.

  • Mean ± standard mean error of three assays.

  • ‡§¶

    Positive control of ChEs.

Fucosterol (1)>500421.72 ± 1.43
24-hydroperoxy 24-vinylcholesterol (2)389.1 ± 2.29176.46 ± 2.51
Phloroglucinol (3)>500>500
Eckstolonol (4)42.66 ± 8.48230.27 ± 3.52
Eckol (5)20.56 ± 5.61>500
Phlorofucofuroeckol-A (6)4.89 ± 2.28136.71 ± 3.33
Dieckol (7)17.11 ± 3.24>500
Triphlorethol-A (8)>500>500
2-phloroeckol (9)38.13 ± 4.95>500
7-phloroeckol (10)21.11 ± 4.16>500
Eserine0.02 ± 0.0020.06 ± 0.002
Tacrine§0.06 ± 0.0040.001 ± 0.0001
Berberine0.06 ± 0.0011.29 ± 0.007

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

Many investigations have previously attempted to develop ChEs inhibitors for the treatment of AD, either synthetically or via the exploitation of plants and fungi used in traditional medicine.57 ChEs inhibitors have been confirmed to show some efficacy in the treatment of AD, but research into an effective agent from marine algae is still in its infancy.

The phlorotannins, compounds 5, 7, 9 and 10, showed selective dose dependent inhibitory activities toward AChE; whereas, compounds 4 and 6 exhibited inhibitory activities toward both AChE and BChE. Neither the monomer, phloroglucinol (3), nor triphlorethol-A (8), the opened-chain trimer of phloroglucinol, inhibited the ChEs assays at the concentrations tested. These differences may be explained by the specific binding properties between the enzyme and substrate: AChE is a substrate specific enzyme, which degrades the neurotransmitter, acetylcholine, in the nerve synapse, while BChE is a non-specific enzyme in the plasma and tissue.58–61 Cholinesterase inhibitors represent the most promising therapeutic agents for Alzheimer's type dementia patients, as shown by the clinical studies on the effects of these drugs on cognition (memory and concentration) and behavioral symptoms (apathy and motor agitation).62 AChE inhibitors boost the endogenous levels of acetylcholine in the brains of Alzheimer's disease patients and; thereby, increase cholinergic neurotransmission. BChE inhibitors decrease the increased number of neuritic plaques in demented brains.63 Therefore, a good balance between the inhibitions of AChE and BChE may result in higher efficacy.63

Phlorotannins are polymers of phloroglucinol; therefore, have appropriately bulky structures, which masks the ChEs and prevents the binding of the substrates (ACh or BCh) in a non-competitive manner (data not shown). In contrast, in the case of triphlorethol-A, which has an open dibenzo-1, 4-dioxine moiety and a bulky structure, the activity may simply be deactivated due to the difficulty in the accessibility to the ChEs. Therefore, it was concluded that the degree of polymerization and closed-ring structure of phlorotannins must play key roles in the inhibitory potential of phlorotannins toward the ChEs.

Phlorofucofuroeckol A (6) and dieckol (7) have previously been reported to exhibit significant AChE inhibitory activities.64 The present study demonstrated that these phlorotannins (compounds 6 and 7) also possess inhibitory activities toward AChE. Since most AChE inhibitors are known to contain nitrogen,7–13 the higher activities of the phlorotannins harbor potential as new AChE inhibitors.

Fucosterol (1) was found to be a selective inhibitor of BChE. Conversely, 24-hydroperoxy 24-vinylcholesterol (2) showed inhibitory activities toward both AChE (IC50: 389.10 ± 2.29 μM) and BChE (IC50: 176.50 ± 5.04 μM). As fucosterol is usually obtained from the non-polar solvent fractions of seaweed extracts, research into the bioactivities of these compounds have generally focused on their antioxidant, antidiabetic and hepatoprotective activites.65,66 The sterols isolated from Haloxylon recurvum have been reported to exhibit significant ChEs inhibitory activities.28

Eserine, used as a positive control, also known as (–)-physostigmine, is a reversible cholinesterase inhibitor isolated from the Calabar bean, which is used to treat a damaged central nervous system. However, its side-effects, including depression and overdose, can cause cholinergic syndrome.67

In conclusion, the ethanolic extract of E. stolonifera has been shown to significantly inhibit the ChEs activities. Various phlorotannins and sterols isolated from the extract are responsible for the ChEs inhibitory activities. Furthermore, the structure-activity relationships of the phlorotannins can provide useful information on the interaction between the ChEs and ligands. The mechanism responsible for the in vitro inhibitory activities of phlorotannins, and whether these compounds have antiamnestic effects, are currently being studied in the authors laboratory.

ACKNOWLEDGMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

This work was supported by a grant from the KOSEF (R01-2005-000-106610).

REFERENCES

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  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES
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