Correlation of branching structure of mushroom β -glucan with its physiological activities

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Correlation of branching structure of mushroom β -glucan with its physiological activities

In Young Bae

^a

, Hye Won Kim

^a

, Hyun Jae Yoo

^a

, Eun Suh Kim

^a

, Suyong Lee

^b

, Dong Yun Park

^c

, Hyeon Gyu Lee

^a,

⁎

aDepartment of Food and Nutrition, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, Republic of Korea

bDepartment of Food Science & Technology and Carbohydrate Bioproduct Research Center, Sejong University, 98 Gunja-dong, Gwangjin-gu, Seoul 143-747, Republic of Korea

cHana Natural Substance Institute, Hanabiotech Ltd., 456 Yoochon, Misanmyun, Yeoncheon-gun, Kyunggi-do, 486-860, Republic of Korea

a b s t r a c t a r t i c l e i n f o

Article history:

Received 29 August 2012 Accepted 5 December 2012 Available online 20 December 2012 Keywords:

Lentinus edodes β-Glucan Degree of branching Cancer cell growth inhibition Nitric oxide production

β-Glucans with a different degree of branching (DB) were prepared from three different mushrooms and their physiological properties were characterized in terms of branching structure.β-Glucan with 29% DB from shitake mushroom was the most effective in inhibiting the growth of tumor cells and also producing nitric oxide compared to Chamsong-I (67% DB) and cauliﬂower (16% DB)β-glucan. In addition, theβ-(1-6)-linked branches ofβ-glucan from Chamsong-I mushroom were speciﬁcally hydrolyzed by enzymatic treatments to produce β-glucan samples with various DB from 19 to 50%. As the DB ofβ-glucan reduced, the inhibition of cancer cell growth and production of nitric oxide increased when the branching reached up to 32%, then decreased as the DB further reduced. Therefore, these results indicated that the branching structure ofβ-glucan plays a crucial role in its antitumor effect and the antitumor effect could be enhanced by modulating critical branching structure ofβ-glucan.

1. Introduction

Mushrooms have been worldwide consumed as a good source of functional ingredients with beneﬁcial health effects. Specially, out of the functional ingredients derived from mushrooms, scientiﬁc and industrial attention has been paid toβ-glucan since it is well known to exhibit a variety of physiological properties including antitumor and immune modulating properties (Lombard, 1994). The physiological effects of mushroomβ-glucan vary widely depending on the structural characteristics, including molecular weight, chemical composition, and chain conformation (Wasser, 2002). Thus, a great deal of effort has been made to characterize the structural and biological properties of mush- roomβ-glucans and also to modify their structure by incorporating new functional groups into the polymer chain (Bae, Kim, Lee, & Lee, 2011; Jung, Bae, Lee, & Lee, 2011; Shin, Lee, Bae, Yoo, & Lee, 2007;

Wang, Zhang, Li, Hou, & Zeng, 2004).

There are several preceding studies that investigated the antitumor activity of mushroom β-glucan regarding its branching structure.

β-Glucan containing mainly (1-6) linkages exhibits weak antitumor activity, compared withβ-(1-6)-branched (1-3)-β-glucan (Mizuno, 2000; Mizuno et al., 1999). Lentinan, the (1-3)-(1-6)-β-D-glucan derived fromLentinus edodes with 20 to 30% branching, has the highest antitumor activity (Sasaki & Takasuka, 1976). In addition,

the proteoglucan fromAgaricus blazeiMurrill fruiting bodies, which consists of α-(1-4)-glucan with β-(1-6)-branching at a ratio of approximately 4:1, exhibits the most potent antitumor capacity (Fujimiya et al., 1998). Thus, it was reported thatβ-(1-3) linkages in the main chain and additionalβ-(1-6) branch points are required for its antitumor action (Demleitner, Kraus, & Franz, 1992; Surenjav, Zhang, Xu, Zhang, & Zeng, 2006). However, no effort has been made to monitor the antitumor activity ofβ-glucan by enzymatically con- trolling its branching point. More extensive studies are needed for better understanding of the relationship betweenβ-glucan structure and antitumor function.

Cauliflower mushroom (Sparassis crispa), one of the three mushrooms used in this study, is known to have beneficial health effects including antitumor activity, immune stimulation, and anti-inflammatory effects (Harada et al., 2002; Kim, Park, Cho, Ko, & Kim, 2009; Ohno, Miura, Nakajima, & Yadomae, 2000). In addition, it is widely recognized as a good source ofβ-glucan (approximately 43.6% of its dry weight) (Ohno et al., 2000). Also, Shiitake mushroom (L. edodes), is regarded as a source of lentinan, which is licensed as an anticancer drug in Japan (Chihara, Himuri, Maeda, Arai, & Fukuoka, 1970). Chamsong-I mushroom (L. edodes) is a newly cultivated mushroom in Korea and originat- ed from Shiitake mushrooms (Bae et al., 2010). While Chamsong-I mushroom is shaped like a pine mushroom (Tricholoma matsutake), it tastes like Shiitake mushroom (Bae et al., 2010). Although the content ofβ-glucan in Chamsong-I mushroom is reported to be four times higher than that in Shiitake mushrooms, the structure and health-benefits of Chamsong-I mushroomβ-glucan have not been fully investigated.

⁎ Corresponding author. Tel.: +82 2 2220 1202; fax: +82 2 2292 1226.

E-mail address:hyeonlee@hanyang.ac.kr(H.G. Lee).

http://dx.doi.org/10.1016/j.foodres.2012.12.008

Contents lists available atSciVerse ScienceDirect

Food Research International

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d r e s

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In this study,β-glucans from three different mushrooms (Chamsong-I, Shiitake, and Cauliﬂower) were extracted and their physiological properties were compared in terms of the degree of branching. In addition, β-glucans from Chamsong-I mushrooms with different degrees of branching were enzymatically prepared and the effect of degree of branching ofβ-glucans on the antitumor activity and nitric oxide production was investigated.

2. Material and method

2.1. Extraction and puriﬁcation of mushroomβ-glucans

Chamsong-I, Shiitake, and Cauliflower mushrooms were kindly provided by Hanabiotech Ltd. (Kyunggi-do, Korea). The mushroom fruiting bodies were air-dried and ground (under 100 mesh) to extract β-glucans. After extraction, the β-glucans were purified according to the method ofCamelini et al. (2005)with slight modifi- cations. Mushroom powder (100 g) was suspended in distilled water (1 L) and the pH was adjusted to 12 with 2 M NaOH, followed by ag- itation at 95 °C for 12 h. After centrifugation at 6500gfor 20 min, three volumes of ethanol were added to the supernatant and left at 4 °C for 24 h, followed by centrifugation at 6500gfor 20 min. The precipitates were re-suspended in distilled water and Sevag treatment (Sevag, Lackman, & Smolens, 1938) was applied to remove proteins. The precipitates were then dialyzed (molecular weight cut-off 10,000 Da, Spectrum Laboratories, Gardena, CA, USA) against distilled water forfive days. The homogeneity of purifiedβ-glucan was confirmed by gel permeation chromatography (GPC) (Agilent 1100 series, Palo Alto, CA, USA) using with BioSep-SEC-S2000 and S4000 columns (300×7.8 mm, Phenomenex, Inc., Torrance, CA, USA) and a refrac- tive index detector.

2.2. Preparation of modifiedβ-glucans byβ-(1-6)-glucanase treatment The structure of β-glucan from Chamsong-I mushroom was modified with β-(1-6)-glucanase (Westase, EC 3.2.1.75, Takara, Tokyo, Japan) according to the methods ofFrieman, McCaffery, and Cormack (2002)andHirokawa et al. (2008). Purified Chamsong-I β-glucan powders (1000μg/mL) andβ-(1-6)-glucanase (20μg/mL) were prepared in 50 mM phosphate buffer (pH 7.4), respectively.

Then, theβ-(1-6)-glucanase solution was added to theβ-glucan dis- persion and incubated for 1, 2, 4, 8, and 12 h at 40 °C in order to pro- duceβ-glucan samples with different degrees of branching. After hydrolysis for each speciﬁed reaction time, the mixture was heated at 100 °C for 10 min to inactivate the enzyme and then centrifuged at 3000gfor 10 min. After the supernatant was freeze-dried, it was stored at−80 °C until further analysis.

To investigate the de-branching patterns of theβ-glucan samples, the amount of glucose in the digested sample (100μL) was determined using a glucose oxidase-peroxidase (GOPOD) reagent (Megazyme International Ireland Ltd., Wicklow, Ireland). The samples were centrifuged at 8000 for 30 min, and the supernatant was treated with GOPOD for 20 min. The absorbance of samples was measured at 510 nm against a reagent blank.

2.3. Chemical and structural characterizations

The molecular weights (MW) of theβ-glucan samples were determined by gel permeation chromatography (GPC), performed at room temperature with an HPLC system (Agilent 1100 series, Palo Alto, CA, USA) equipped with BioSep-SEC-S2000 and S4000 columns (300 × 7.8 mm, Phenomenex, Inc., Torrance, CA, USA) and a refrac- tive index detector. Deionized water was used as eluent at aﬂow rate of 1.0 mL/min, the injection volume (0.1%, w/v) was 0.1 mL, and dextran standards with known MW values were used.

Monosaccharide composition was measured using liquid chromatography (HPAEC-PAD system, Dionex DX500, Sunnyvale, CA, USA) with a Carbo-Pac™PA1 (4 ×250 mm) column.β-Glucan samples were hydrolyzed with triﬂuoroacetic acid at 100 °C for 4 h and a 20μL aliquot of the sample was injected. Eluent andﬂow rate were 18 mM NaOH solution and 1.0 mL/min, respectively.

β-Glucans were methylated according to the procedure ofXu, Xu, and Zhang (2012),Shin, Kiyohara, Matsumoto, and Yamada (1997), andHakomori (1964). The resulting methylated samples were then subjected to acetylation using acetic anhydride. Methylated alditol acetate was dissolved in dichloromethane, extracted with distilled water several times, and then the organic layer was analyzed using a Hewlett-Packard 5890A GC system (Tokyo, Japan) equipped with an SP-2380 capillary column (0.25 mm × 30 m, 0.2 umﬁlm thick- ness, Supelco Inc., Bellefonte, PA, USA). The column temperature was programmed at 60 °C for 1 min, 60→150 °C (30 °C/min), 150→180 °C (1 °C/min), 180→231 °C (1.5 °C/min), 231→250 °C (30 °C/min), and 250 °C for 10 min. Helium was used as a carrier gas (0.5 mL/min) and a 5970 mass selective detector was used to identify the fragmented ions. The degree of branching (DB) was cal- culated based on the following formula (Hawker, Lee, & Frechet, 1991; Tao & Zhang, 2006):

Degree of branching DBð Þ ¼ðN_TþN_BÞ=ðN_TþN_BþN_LÞ

where N_T, N_B, and N_Lare the total mol% of terminal residues, branched residues, and linear residues, respectively.

2.4. Cancer cell growth inhibition

The cytotoxic activities of theβ-glucan samples against MCF-7 breast cancer cells and Sarcoma 180 cells (Korean Cell Line Bank, Seoul, Korea) were investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich Chemical Co.). The cells were cultured in RPMI1640 medium (JBI Welgene Co., Daegu, Korea) supplemented with 10% fetal bovine serum and 100 units/mL penicillin/streptomycin solution (Sigma-Aldrich Chemical Co.).β-Glucan solutions at various concentrations (native β-glucans extracted from mushrooms, 10, 50, 100 and 200μg/mL;

enzymatically-modifiedβ-glucans, 10, 25, 50, 100 and 200μg/mL) were added to the cells (1 × 10⁴cells/well) in 96-well tissue culture plates (Greiner) and incubated in a 5% CO₂ incubator (37 °C) for 92 h. MTT was then added to each experimental well and incubated at 37 °C for an additional 4 h. Then, 150μL DMSO (Sigma-Aldrich Chemical Co.) was added to solubilize the formazan crystals, and the plates were agitated on a plate shaker for 15 min. The optical density of each well was measured at 540 nm using a multi-well ELISA automatic spectrometer reader (ELx800UV, Bio-Tek Instru- ment Inc., Windoski, VT, USA). The well-known anti-cancer agent, 5-fluorouracil (5-FU, Sigma-Aldrich Chemical Co.), was used as a positive control at afinal concentration of 5μg/mL.

2.5. Nitric oxide production

RAW 264.7 cells were obtained from the Korean Cell Line Bank and used to investigate nitric oxide production. A cell suspension (5.0 × 10⁵cells/mL) was prepared in Dulbecco's modiﬁed Eagle's medium (DMEM) (Cambrex Bio Science Walkersville Inc., Walkersville, MD, USA) with 10% fetal bovine serum and 100 units/mL penicillin/

streptomycin (Sigma-Aldrich Chemical Co., Milwaukee, WI, USA), and 1 mL of this suspension was transferred to 12-well tissue culture plates (Greiner, Nurtingen, Germany). After incubating at 37 °C for 2 h in 5% CO2, the medium was changed with DMEM containing the sample solutions (nativeβ-glucans extracted from mushrooms, 10–100μg/mL; enzymatically-modiﬁedβ-glucans, 100μg/mL), and then incubated for an additional 24 h. The supernatant was treated

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with an equal volume of Griess reagent (1% sulfanilamide, 0.1%

naphthalene diamine dihydrochloride, 2.5% H3PO4) (Sigma-Aldrich Chemical Co.) at room temperature for 2 min. Nitric oxide generation was estimated by measuring the optical density at 540 nm against sodium nitrite as a standard. Lipopolysaccharide (LPS from Salmonella entericaserotype Typhimurium, Sigma-Aldrich Chemical Co.) was used as a positive control at a ﬁnal concentration of 0.1μg/mL.

2.6. Statistical analysis

All assays were performed in triplicate. The experimental data were analyzed by one-way ANOVA using SPSS 12.0 (Statistical Package for Social Science, SPSS Inc., Chicago, IL, USA). Signiﬁcant differences between samples were estimated using Duncan's multiple range tests at a p-value of 0.05.

3. Results and discussion

3.1. Chemical and structural characterization of mushroomβ-glucans The average molecular weights of puriﬁed β-glucans from Chamsong-I, Shiitake, and Cauliﬂower mushrooms were determined to be 280, 440, and 330 kDa, respectively (Table 1). The results of monosaccharide composition showed that glucose was the major monosaccharide for all three mushroomβ-glucans and the glucose content ranged from 77 to 94%. The residues of galactose (4–12%), mannose (6–7%), and fucose (2–3%) were also found.

The linkage type of methylatedβ-glucans was determined by GC–MS analysis and the components corresponding to the individual peaks were identiﬁed by the relative abundance of fragmented ion peaks in the mass spectra. The most abundant products were 2,4,6-tri-O- methylglucose (1→3 linked glucose), 2,3,4-tri-O-methylglucose (1→6 linked glucose), 2,4-di-O-methylglucose (1→3 and 1→6 linked glucose), and 2,3,4,6-tetra-O-methylglucose (terminal glucose) (Table 2). Based on these results,β-glucans extracted from Chamsong-I, Shiitake, and Cauli- ﬂower mushrooms were composed of (1-3)-β-glucans with (1-6)-linked branches.

When the branch structures ofβ-glucans from three mushrooms were analyzed by GC–MS, the ratios of NT, NB, and NLin theβ-glucans from Chamsong-I, Shiitake, and Cauliﬂower mushrooms were 20.1:46.9:(24.5+ 8.5), 18.9:10.3:(65.2 + 5.6), and 8.5:7.3:(77.4+ 6.9),

respectively. Thus, the degree of branching (DB) was determined to be 67, 29, and 16% for Chamsong-I, Shiitake, and Cauliﬂower mushroom β-glucans, respectively. Since the Chamsong-Iβ-glucan had the highest DB value, it was chosen to enzymatically produceβ-glucan samples with different degrees of branching. This was discussed in the later section of this study.

3.2. Effect of branch structure on the antitumor activity ofβ-glucans β-Glucan samples from three mushrooms at a dosage level of 10–200μg/mL caused a dose-dependent repression of cell proliferation with an inhibition range of 7.2–55.1% and 6.2–53.3% on MCF-7 and Sar- coma 180 cells, respectively (Fig. 1). In particular, the mushroom- derivedβ-glucans at 200μg/mL were more effective in suppressing

Table 1

Molecular weight and monosaccharide composition of β-glucans extracted from Chamsong-I, Shitake, and Cauliﬂower mushrooms.

Mushroom MW^a(kDa) Monosaccharide composition^b(area %)

Glc Fuc Man Gal

Chamsong-I 280 80.1 3.0 5.5 11.5

Shiitake 440 77.0 3.3 6.8 12.2

Cauliﬂower 330 94.4 1.9 – 3.5

a MW, average molecular weight.

b Glc, glucose; Fuc, fucose; Man, mannose; and Gal, galactose.

Table 2

GC–MS data for the methylation analysis ofβ-glucans extracted from Chamsong-I, Shitake, and Cauliﬂower mushrooms.

Mushroom Degree of branching (%) Linkage type^a(%)

β-1,3 β-1,6 β-1,3,6 Terminal

Chamsong-I 67.03 ± 7.8 24.49 8.48 46.92 20.10

Shiitake 29.17 ± 3.3 65.23 5.60 10.27 18.91

Cauliﬂower 15.54 ± 3.8 77.37 6.87 7.28 8.48

a β-1,3-linkage, 2,4,6-tri-O-methylglucose; β-1,6-linkage, 2,3,4-tri-O-methylglucose; β-1,3,6-linkage, 2,4-di-O-methylglucose; and terminal, 2,3,4,6-tetra-O-methylglucose.

Fig. 1.Cancer cell growth inhibition ofβ-glucans extracted from Chamsong-I (a), Shitake (b), and Cauliflower (c) mushrooms against MCF-7 and Sarcoma 180 cells. Different letters on the bars indicate a significant difference at the 5% level. 5-FU: 5-fluorouracil (5μg/mL). All the values are mean±SD (n=3).

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MCF-7 cells than Sarcoma 180 cells, yielding similar values to the activity of 5-FU. It might be caused by cytotoxic speciﬁcity for particular tumor cells (Shin et al., 2007). These results were also similar to those of previous reports which showed that a family ofL. edodesglucans consisting of (1-3)-β-D-glucan backbones with (1-6)-β-D-glucose exhibited immune-mediated antitumor activity (Wang, Zhang, Zhang, Zhang, & Li, 2009; Zhang, Li, Xu, & Zeng, 2005).

Nitric oxide secreted by activated macrophages is known to pro- tect the host against infectious invaders (Huffman et al., 2003). In addition,β-glucan binds speciﬁc receptors on macrophages to activate them, resulting in the release of nitric oxide (Jung et al., 2004). There- fore, the production of nitric oxide in macrophages contributes to the inhibition of cancer cell proliferation, thus enhancing the antitumor activity ofβ-glucan.

Fig. 2exhibits the effect of mushroomβ-glucans on the production of nitric oxide. Allβ-glucan samples from Chamsong-I, Shiitake, and Cauliflower mushrooms generated nitric oxide in a concentration- Fig. 2.Production of nitric oxide by RAW264.7 cells treated withβ-glucans extracted from Chamsong-I, Shitake, and Cauliflower mushrooms. Different letters on the bars indicate a significant difference at the 5% level. LPS: Lipopolysaccharide fromSalmonella entericaserotype Typhimurium (0.1μg/mL). All the values are mean ± SD (n = 3).

Fig. 3.Effects of degree of branching of variousβ-glucans on cancer cell growth inhibition (a) and nitric oxide production (b). Different letters on the bars indicate a signiﬁcant difference at the 5% level. All the values are mean±SD (n=3).

Fig. 4.De-branching curves of aβ-(1-6) glucanase treatment (a) and degree of branching (b) of enzyme-modiﬁedβ-glucans from Chamsong-I mushroom. All the values are mean ± SD (n = 3).

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dependent manner, producing 9.5-50.5, 7.4–17.7, and 9.3–16.5μM nitric oxide, respectively. Speciﬁcally, at a concentration of 100μg/mL, Shiitake-derivedβ-glucan produced 5.2-fold more nitric oxide than LPS (9.8μM at 0.1μg/mL).

Although allβ-glucans tested in this study exhibited dose-dependent antitumor activities regardless of origin, the degree of antitumor activity differed among the samples. Specially, when the antitumor effect of β-glucan was correlated to its branch structure (Fig. 3), Shitake β-glucan with 29% DB was the most effective in producing nitric oxide and inhibiting cell growth. This could be favorably compared to the previous results bySasaki and Takasuka (1976)that reported that the highest antitumor activity of Shitakeβ-glucan was observed at around 30% DB. This was probably related to the speciﬁc structural properties ofβ-glucans including 20–30% DB and a triple-helical structure. Thus, in order to conﬁrm the relationship between β-glucan branching degree and antitumor effect,β-glucans with different degrees of branching were enzymatically prepared from Chamsong-I mushroomβ-glucan and their antitumor activities were characterized.

3.3. Antitumor activity ofβ-glucan as a function of de-branching degree Fig. 4 shows the hydrolysis pattern and degree of branching of Chamsong-I mushroomβ-glucan when treated withβ-(1-6)-glucanase

for 48 h. The degree of branching sharply decreased in the initial stages of the reaction, gradually decreased for 8 h, and then reached a plateau.

Thus, it would appear that theβ-(1-6)-branch linkage ofβ-glucan was digested within 12 h. Based on these results, theβ-glucan samples hydrolyzed for 1, 4, 8, and 12 h were collected and then subjected to methylation, followed by GC–MS analysis for the determination of their DBs.

As can be seen inFig. 4(b), the untreatedβ-glucan was linked by 67%

branching. The DB of the enzymatically-modiﬁedβ-glucans decreased with increasing hydrolysis time, showing 19, 24, 32, and 50% branching.

Thus, the modiﬁedβ-glucans with various DBs were successfully prepared and their antitumor activities were compared to those of the control with 67%.

All of the native and de-branchedβ-glucans caused the growth inhibition of MCF-7 or Sarcoma 180 cells in a concentration-dependent manner (Fig. 5). However, the growth inhibition of cancer cells tended to increase with decreasing DB up to 32% at the same dosage level ofβ-glucan (Fig. 6(a)). Moreover, the degree of cancer cell growth retardation dropped sharply as the branching decreased further from 32 to 19%. As shown inFig. 6(b), the nitric oxide generation in RAW264.7 cells tended to increase with decreasing DB up to 32% at the same level ofβ-glucan dosage. These antitumor activities showed a similar pattern to the results shown inFig. 3.

Yoon, Yoo, Cha, and Lee (2004)reported that the branching structure of levan played a crucial role in expressing antitumor activity through reduced cytotoxic effects against cancer cell lines with increasing degree of de-branching. There are speciﬁc receptors forβ-glucan on the surface of macrophages (Borchers, Keen, & Gershwin, 2004). Thus, β-glucan with around 30% DB may optimize the interactions with the macrophage receptors, thereby enhancing the release of nitric oxide and contributing to the antitumor activity.

4. Conclusion

The degree of branching (DB) ofβ-glucan from Chamsong-I mushrooms was enzymatically controlled and the relation between DB of β-glucan and its antitumor activity was investigated. When DB was

Fig. 5.Cancer cell growth inhibition of enzyme-modiﬁedβ-glucans from Chamsong-I mushrooms against MCF-7 (a) and Sacorma 180 (b) cells. All the values are mean±SD (n=3).

Fig. 6.Changes in cancer cell growth inhibition (a) and nitric oxide production (b) of enzyme-modiﬁedβ-glucans. Different letters on the lines indicate a signiﬁcant difference at the 5% level. All the values are mean±SD (n=3).

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reduced to around 32%, the inhibitory activities against MCF-7 human breast cancer cells and Sarcoma 180 tumor cells were improved and the generation of nitric oxide was also enhanced. Thus, these results conﬁrmed the relationship between branch structure of mushroom β-glucan and its antitumor effects, suggesting that there is a speciﬁc degree of branching ofβ-glucan for enhancing its antitumor activity.

Acknowledgments

This work was supported by Mid-career Researcher Program through NRF grant funded by the MEST (2011-0000197).

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