Journal of Ethnopharmacology 121 (2009) 372378

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Identication of Escherichia coli enterotoxin inhibitors from traditional medicinal herbs by in silico, in vitro, and in vivo analyses
Jaw-Chyun Chen a,1,2 , Tin-Yun Ho b,1 , Yuan-Shiun Chang a , Shih-Lu Wu c , Chia-Cheng Li b , Chien-Yun Hsiang d,
a

Graduate Institute of Chinese Pharmaceutical Sciences, China Medical University, Taichung, Taiwan Molecular Biology Laboratory, Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan Department of Biochemistry, China Medical University, Taichung, Taiwan d Department of Microbiology, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan
b c

a r t i c l e

i n f o

a b s t r a c t
Ethnopharmacological relevance: Glycyrrhiza uralensis has been used for the treatment of gastrointestinal disorders, such as diarrhea, in several ancient cultures. Glycyrrhizin is the principal component of liquorice and lots of pharmacological effects have been demonstrated. Aim of the study: Heat-labile enterotoxin (LT), the virulence factor of enterotoxigenic Escherichia coli, induces diarrhea by initially binding to the GM1 on the surfaces of intestinal epithelial cells and consequently leading to the massive loss of uid and ions from cells. Therefore, we evaluated the inhibitory effects of traditional medicinal herbs (TMH) on the B subunit of LT (LTB) and GM1 interaction. Materials and methods: The inhibitory effects of TMH on LTB-GM1 interaction were evaluated by GM1 enzyme-linked immunosorbent assay (ELISA). The likely active phytochemicals of these TMH were then predicted by in silico model (docking) and analyzed by in vitro (GM1 -ELISA) and in vivo (patent mouse gut assay) models. Results: We found that various TMH, which have been ethnomedically used for the treatment of diarrhea, inhibited the LTB-GM1 interaction. Docking data showed that triterpenoids were the most active phytochemicals and the oleanane-type triterpenoids presented better LTB-binding abilities than other types of triterpenoids. Moreover, by in vitro and in vivo models, we demonstrated that glycyrrhizin was the most effective oleanane-type triterpenoid that signicantly suppressed both the LTB-binding ability (IC50 = 3.26 0.17 mM) and the LT-induced uid accumulation in mice. Conclusions: We found an LT inhibitor, glycyrrhizin, from TMH by in silico, in vitro, and in vivo analyses. 2008 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 27 December 2007 Received in revised form 3 November 2008 Accepted 8 November 2008 Available online 21 November 2008 Keywords: Docking analysis Heat-labile enterotoxin Glycyrrhizin GM1

1. Introduction Diarrheal disease remains a leading global health problem. Enterotoxigenic Escherichia coli (ETEC) is the most important pathogen of diarrhea in infants, children, and adults, accounting for 280 million episodes and more than 400,000 deaths annually (WHO, 2005). ETEC is also the most common pathogen of travelers

Abbreviations: E. coli, Escherichia coli; ETEC, Enterotoxigenic Escherichia coli; GM1 ELISA, GM1 -enzyme-linked immunosorbent assay; LT, heat-labile enterotoxin; LTA, A subunit A of LT; LTB, B subunit of LT; PBS, phosphate-buffered saline; TMH, traditional medicinal herbs. Corresponding author. Tel.: +886 4 22053366x2163; fax: +886 4 22053764. E-mail address: cyhsiang@mail.cmu.edu.tw (C.-Y. Hsiang). 1 These authors contributed equally to this work. 2 Present address: Department of Microbiology, China Medical University, Taichung, Taiwan. 0378-8741/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.11.011

diarrhea that affects 10 million travelers in developing countries (Aranda-Michel and Giannella, 1999). Heat-labile enterotoxin (LT) is the major virulent factor of ETEC (Holmgren and Svennerholm, 1992). LT comprises of one A subunit and ve identical B subunits (Merritt and Hol, 1995). The mechanism of diarrhea induced by LT is initiated by the binding of B subunits (LTB) to the ganglioside GM1 [Gal 1-3GalNAc 1-4 (Neu5Ac 2-3)Gal- 1-4Glc-Ceramide] on the surfaces of intestinal epithelial cells (Spangler, 1992; Pickens et al., 2002). Binding of LTB to GM1 induces a conformational change in the toxin molecule, followed by the translocation of A subunit (LTA) into the cells. Inside the intestinal cells, LTA catalyzes the ADP-ribosylation of the stimulatory GTP-binding protein, resulting in the increased intracellular levels of cyclic AMP. Elevated levels of cyclic AMP in the cells cause massive loss of uid and ions from the cells, consequently leading to the symptom of diarrhea (Spangler, 1992). Therefore, the binding of LTB to GM1 is an attractive target for developing drugs or pro-

J.-C. Chen et al. / Journal of Ethnopharmacology 121 (2009) 372378 Table 1 The inhibitory abilities of TMH, which have ethnomedically been used for the treatment of diarrhea. Herbal name Plumula Nelumbinis Cortex Cinnamomi Rhizome Coptis Radix Glycyrrhizae Flos Caryophylli Cortex Magnoliae Ofcinalis Radix Angelicae Sinensis Radix Bupleuri Rhizoma Alismatis Radix Ginseng Latin name Nelumbo nucifera Cinnamomon cassia Coptis chinensis Glycyrrhiza uralensis Eugenia caryophyllata Magnoliae ofcinalis Angelica sinensis Bupleurum chinense Alisma orientalis Panax ginseng Family Nymphaeaceae Lauraceae Ranunculaceae Leguminosae Myrtaceae Magnoliaceae Umbelliferae Umbelliferae Alismataceae Araliaceae

373

Inhibitory ability (%) 79.3 76.3 73.7 73.3 72.9 71.8 71.4 71.1 69.5 65.0

phylactics for the treatment and prevention of LT-induced diarrhea (Minke et al., 1999a; Pickens et al., 2002; Mitchell et al., 2004). Many traditional medicines and their components have been used to treat LT-induced diarrhea. Some herbal extracts, such as the fruit of Chaenomeles speciosa (Chen et al., 2007a), the root of Zingiber ofcinale (Chen et al., 2007b), the leaf of Camellia japonica (Bruins et al., 2006) and the gall on Rhus chinensis (Chen et al., 2006), exhibit anti-LT-induced diarrheal abilities via several mechanisms. Traditional medicinal herbs (TMH) have been used in the treatment of gastrointestinal diseases in China for millennia. Therefore, we collected TMH, which have been ethnomedically used for gastrointestinal disorders, in this study. By GM1 -enzymelinked immunosorbent assay (ELISA), we found that some of TMH extracts were capable of inhibiting the binding of LT to GM1 . The likely active phytochemicals of these TMH were then predicted by in silico model (docking) and analyzed by both in vitro (GM1 -ELISA) and in vivo (patent mouse gut assay) models. Our data showed that glycyrrhizin exhibited anti-diarrheal effects in mice via the inhibition of GM1 and LTB interaction. These ndings suggested that glycyrrhizin might be a potent candidate for the treatment of LTinduced diarrhea. 2. Materials and methods 2.1. Chemicals and herbal materials TMH were gifts from Sun Ten Pharmaceutical Co., Ltd. (Taipei, Taiwan), a renowned GMP manufacturer of concentrated herbal extracts by both Taiwanese and Australian authorities. The qualities of TMH samples were veried by authors (Prof. Yuan-Shiun Chang and Dr. Jaw-Chyun Chen) according to the Pharmacopoeia Commission of Peoples Republic of China (2005). The voucher specimens were deposited in the Molecular Biology Laboratory, Graduate Institute of Chinese Medical Science, China Medical University. Specimen number was listed in supplemental table. TMH sample was ground with the homogenizer to a ne powder and extracted by mixing 100 g of herb powder with methanol at 4 C overnight. The supernatant was then collected and stored at 30 C in small aliquots. Glycyrrhizin was purchased from Sigma (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide at 100 mM. 2.2. Expression and purication of Escherichia coli (E. coli) LT and LTB Recombinant LT and LTB were expressed in E. coli BL21(DE3)pLysS strain and puried by afnity chromatography as described previously (Chen et al., 2006). Briey, cells were induced by 0.5 mM isopropyl- -d-thiogalactopyranoside and collected 3 h after induction. The cell pellet was resuspended in 1 TEAN buffer (50 mM TrisHCl, pH 7.5, 1 mM EDTA, 200 mM NaCl, 3 mM NaN3 ), lysed by sonication, and centrifuged at 15,000 g for 20 min at 4 C. The supernatant was collected and mixed with

d-galactose resin (Pierce, Rockford, IL, USA), and the recombinant LT and LTB were then eluted by 1 TEAN buffer containing 1 M galactose. Proteins were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and quantied with a Bradford assay (Bio-Rad, Hercules, CA, USA). 2.3. Competitive GM1 -ELISA LTB was biotinylated as described previously (Chen et al., 2006). Biotinylated LTB (16 ng) was mixed with various amounts of compounds and incubated at 4 C for 3 h with shaking. Microtiter plates (MaxiSorp Nunc-ImmumTM plates, Nunc, Denmark) were coated with 100 l of 2 ng/ l GM1 (Sigma, St. Louis, MO, USA), which was diluted in phosphate-buffered saline (PBS) (137 mM NaCl, 1.4 mM KH2 PO4 , 4.3 mM Na2 HPO4 , 2.7 mM KCl, pH 7.2), per well and incubated at 4 C overnight. The wells were washed with 200 l of washing buffer (0.5% Tween 20 in PBS), blocked with 200 l of blocking buffer (1% bovine serum albumin in PBS) at 37 C for 1 h, and then incubated with 100 l of biotinylated LTB/compound mixture at 37 C for 1 h. After three washes with washing buffer, 50 l of diluted peroxidase-conjugated avidin (Pierce, Rockford, IL, USA) was added to each well and incubated at 37 C for 1 h. Following

Fig. 1. Inhibitory abilities of TMH on the interaction between LTB and GM1 . Numbers in the brackets are the sum of herb species in the family. * p < 0.05, ** p < 0.01, compared with LTB.

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Fig. 2. The predicted binding energy scores of phytochemicals. (A) Average binding energy scores of 600 phytochemicals belonging to 12 different kinds of structures. (B) Average binding energy scores of 8 different types of triterpenoids. (C) Skeletons of 7 different types of triterpenoids.

three washes, 50 l of chromogenic substrate, 2,2 -azinobis(3ethylbenzthiazoline-sulfonic acid) (Sigma, St. Louis, MO, USA), was added to each well and incubated at 37 C for 15 min. The absorbance was read at 405 nm in an ELISA plate reader. The inhibitory ability (%) was calculated by [1 (OD value of mixture containing LTB and compound/OD value of mixture containing LTB only)] 100. 2.4. Docking technology The docking technology was performed as described previously (Chen et al., 2007). The MEDock (Maximum Entropy based Docking) web server (http://medock.csie.ntu.edu.tw/) was used for the prediction of ligand binding sites (Chang et al., 2005). The input le was in the PDBQ format, which is an extension of the PDB format. The PDBQ format of ligands (phytochemicals) was generated by Dundees PRODRG server (http://davapc1.bioch.dundee.ac.uk/programs/prodrg/) (Schttelkopf and van Aalten, 2004). The PDB le (PDB code 1EFI) of LTB was taken from the Protein Data Bank (http://www.rcsb.org/pdb/) (Sixma et al., 1993; Fan et al., 2001).

The PDBQ le of LTB was derived from the PDB2PQR server (http://agave.wustl.edu/pdb2pqr/) (Dolinsky et al., 2004). 2.5. Patent mouse gut assay Female BALB/c mice (8 weeks old, 20 1 g weight) were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Mouse experiments were conducted under ethics approval from the China Medical University Animal Ethics Committee (Ethics Approval Number 96-42-N). In vivo LT-induced diarrheal ability was determined by the patent mouse gut assay as described previously (Baselski et al., 1997; Chen et al., 2006). Briey, ve mice per group were starved with water only for 16 h. Each mouse was inoculated intragastrically with 0.5 ml of 10 g LT alone or in conjunction with various amounts of TMH extracts or compounds. Six hours latter, mice were sacriced. The entire intestine from duodenum to rectum was carefully removed to retain any accumulated uid, and the residual mesentery was removed prior to weigh. The carcass was weighed separately. LT-induced diarrheal ability was presented as a gut/carcass ratio as followed. The IC50 value of compound was

J.-C. Chen et al. / Journal of Ethnopharmacology 121 (2009) 372378 Table 2 Binding energy scores of oleanane type of triterpenoids. Name Structure

375

Energy score (kcal/mol)

Glycyrrhizin

18.03

Saikosaponin A

14.12

Friedelin

11.01

-Hederin

10.41

-Amyrin

8.45

determined as the quantity of compound required to inhibit the LT-induced gut/carcass ratio at 50%. Gut/Carcass ratio = Gut weight(g) Carcass weight(g)

2.6. Statistical analysis Data were presented as mean S.E. Students t-test was used for a comparison between two experiments. A value of p < 0.05 was considered statistically signicant.

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Fig. 4. Anti-diarrheal effect of glycyrrhizin by patent mouse gut assay. Values are mean S.E. of triplicate assays. * p < 0.05, compared with LT treatment. Fig. 3. Inhibitory ability of glycyrrhizin on the binding of LTB to GM1 by competitive GM1 -ELISA. Values are mean S.E. of triplicate assays. ** p < 0.01, compared with LTB treatment.

3.4. Docking analysis of the interaction between glycyrrhizin and LTB We further interpreted the binding sites of glycyrrhizin in LTB by docking technology. Glycyrrhizin was capable of docking into the LTB (Fig. 5). Glycyrrhizin tted LTB very well, with the predicted binding energy score of 18.03 kcal/mol. Hydrogen bonds were formed directly between the carboxylic ketone group of glycyrrhizin and Asn90 residue of LTB. Extra hydrogen bonds were

3. Results 3.1. The inhibitory abilities of TMH on the LTB and GM1 interaction By GM1 -ELISA, we found that various TMH extracts, which have ethnomedically been used for diarrheal disease, blocked the binding of LTB to GM1 (Table 1). We further divided these TMH into different taxonomic families, and their inhibitory abilities were shown in Fig. 1. In a total of 28 families, 8 families, including Campanulaceae, Convolvulaceae, Labiatae, Oleaceae, Polygonaceae, Rosaceae, Solanaceae and Umbelliferae, exhibited approximately 60% inhibitions at 1 g/ l.

3.2. Docking analysis of the interaction between phytochemicals and LTB We further analyzed the representative phytochemicals of these eight families by docking technology. The 3D structures of about 600 phytochemicals, which belong to 12 different kinds of structures (including monoterpenes, sesquiterpenes, iridoids, diterpenes, triterpenoids, alkaloids, quinones, avonoids, tannins, phenylpropanoids, sterols, and others), were constructed and the interactions between phytochemicals and LTB were evaluated by docking analysis. Fig. 2A shows that several kinds of phytochemicals directly docked into the LTB. Triterpenoids were the most active phytochemicals and its average binding energy score was 10.15 1.74 kcal/mol. By further analyzing the phytochemicals belonging to triterpenes, we found that oleanane-type triterpenoids presented better binding abilities than other triterpenoids, and its average binding energy score was 11.14 2.21 kcal/mol (Fig. 2b). The docking data from part of oleanane-type triterpenoids were shown in Table 2. As shown in Table 2, glycyrrhizin exhibited the lowest binding energy score among the oleanane-type triterpenoids and its binding energy score was 18.03 kcal/mol.

3.3. Glycyrrhizin exhibited the anti-LT-induced diarrheal ability by blocking the binding of LTB to GM1 We further analyzed the anti-diarrheal effects of glycyrrhizin by competitive GM1 -ELISA and patent mouse gut assay. Glycyrrhizin signicantly blocked the binding of LTB to GM1 , with the IC50 value of 3.26 0.17 mM (Fig. 3). It also signicantly suppressed the LT-induced uid accumulation at 10 mM (Fig. 4). Therefore, these ndings indicated that the glycyrrhizin was the likely active component for the suppression of LT-induced diarrhea.

Fig. 5. Docking structure between LT and glycyrrhizin. (A) Surface representation of LTB complexes with glycyrrhizin. (B) Close up view of LTB complexes with glycyrrhizin. Glycyrrhizin is represented by stick style. Side chains of selected residues are represented by red wire style. Hydrogen bonds between LTB and glycyrrhizin are represented by green dash lines.

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formed between the hydroxyl groups of glucuronic acids and the Gln49 , Glu51 , Val52 , Gln56 , Thr92 , and Pro93 residues of LTB. Moreover, skeleton of oleanane and the oxane of glucuronic acids exhibited hydrophobic contacts with the Asn14 , Arg35 , Pro53 , Gly54 , Ser55 , His57 , Trp88 , and Lys91 residues as well as with the peptide backbone of LTB. 4. Discussion Antibiotics and antimotility agents are used to control diarrheal symptom. Antibiotics kill bacteria; however, they cannot inhibit the toxicity of bacterial toxin. Moreover, antibiotic therapy is not a viable solution because of the rapid increase of antibiotic resistance, particularly in endemic areas (Garg et al., 2000). Antimotility agents like loperamide can lessen stool frequency and volume in mild diarrhea. However, such agents are not suitable in severe diarrhea because they may cause pooling of large amounts of uid in paralyzed bowel loops (Field, 2003). Additionally, when loperamide is administrated in patients with moderately or severely dehydrated illness or bloody diarrhea, and in patients younger than 3 years old, the adverse effects of loperamide would outweigh benets (Li et al., 2007). The most commonly principle of management for serious diarrhea is the uid replacement combined with pharmacologic therapy (Ahlquist and Camilleri, 2001). Fluid and electrolyte replacement is used to replace uid losses by the administration of sugar-electrolyte solutions; however, it does not facilitate the re-adsorption of secreted uid and therefore does not lessen diarrhea. Because there is currently no specic prophylaxis against diarrhea caused by bacterial toxin, the discovery of agents that can block the function of toxin is important for the prevention and treatment of bacterial toxin-induced diarrhea. To develop the agents that inhibit the toxin-induced diarrhea with specicity, we set up the LT- instead of castor oil-induced diarrheal model. Castor oil is experimentally used for the induction of diarrhea in animal model (Mascolo et al., 1992, 1993). However, the mechanism of castor oil-induced diarrhea is totally different from LT-induced diarrhea. Castor oil administration stimulates the liberation of ricinoleic acid from castor oil, results in the irritation and inammation of the intestinal mucosa, and consequently leads to the release of prostaglandin, which in turn stimulates the intestinal motility and secretion (Pierce et al., 1971; Chitme et al., 2004). The uid accumulation assay and patent mouse gut assay are frequently used as the toxin-induced diarrhea models (Gorbach et al., 1971; Hitotsubashi et al., 1992; Guidry et al., 1997). We used the patent mouse gut assay as the diarrheal model in this study because the induction of diarrhea is through the oral instead of intraintestinal administration of LT. The phytochemicals derived from medicinal plants are abundant sources of biological active compounds. Many phytochemicals have been used as the basis for the development of new lead chemicals for pharmaceuticals (van Der et al., 2004). For examples, quinine and artemisinin, isolated from the Cinchona bark and Artemisia annua, respectively, are the most important lead compounds against malaria (Wright, 2005). Because of increasing reliance on newer technologies, such as in silico analysis models and high-throughput screening, and their associated approaches for drug discovery, natural-product-based drug discovery has successfully and rapidly integrated rational approaches that exploit and evolve the structural diversity provided by nature (Haustedt et al., 2006). Therefore, in silico screening has shown a great promise in drug discovery because it plays an important role in digging out lead (active) compounds from traditional medicine (Shen et al., 2003). In this study, we set up a 3D structure library of 600 phytochemicals from effective TMH and predicted the anti-toxin-binding abilities of phytochemicals by docking analysis. The docking results indi-

cated that several kinds of phytochemicals docked into the binding site of LTB. For examples, Bupleurum chinense, Alisma orientalis, and Panax ginseng extracts blocked the binding of LTB to GM1 , with the inhibitory abilities of 71.1, 69.5, and 65.0%, respectively. Their major triterpenoids Saikosaponin A (oleanane type), Alisol A (protostane type), and ginsenoside Rg2 (dammarane type) also docked into the active site of LTB, with the binding energy scores of 14.12, 10.29, and 9.99 kcal/mol, respectively. Additionally, we found that oleanane-type triterpenoids were the most effective triterpenoids in these families, except Convolvulaceae and Solanaceae (Vincken et al., 2007). Oleanane-type triterpenoids are widely distributed in many TMH and exhibit biological and pharmacological effects (Sparg et al., 2004). Many oleanane-type triterpenoids, such as -amyrin, -hederin and friedelin, docked into the active site of LTB and presented different binding energy scores (8.45, 10.41, and 11.01 kcal/mol, respectively). Our previous study also demonstrated that three kinds of triterpenoids, including oleananic acid, ursolic acid and betulinic acid, dock into the active site of LTB (Chen et al., 2007a). Their binding inhibitory abilities are concentration dependent, with the IC50 value of 202.8 47.8, 493.6 100.0, and 480.5 56.9 M, respectively. These results indicated that oleananic acid (oleanane type) was more active than ursolic acid (ursane type) and betulinic acid (lupane type) in the suppression of LTB and GM1 interaction. Therefore, we suggested that oleanane-type triterpenoids presented better abilities than other types of triterpenoids in the inhibition of LTB and GM1 interaction. Liquorice (radix Glycyrrhiza) is the root of several Glycyrrhiza species. Glycyrrhiza uralensis is one of the major species used in China. Liquorice has been used in China and Europe for thousands of years. It has been used for healing respiratory, gastrointestinal, cardiovascular, and genital-urinary disorders, such as asthma, cough, gastroenteritis, diarrhea, angina, and kidney stones in several ancient cultures (ChPC, 2005; Fiore et al., 2005). Glycyrrhizin is the principal component of liquorice and lots of biological, pharmacological, and toxicity effects have been demonstrated (Blumenthal et al., 2000; Wang and Nixon, 2001). For examples, glycyrrhizin exhibit anti-ulcer, anti-viral, and hepatoprotective effects. In this study, we rst demonstrated that Glycyrrhiza uralensis extract and glycyrrhizin reduced the binding of LTB to GM1 , with the inhibitory abilities of 73.3 and 97.5%, respectively. Moreover, glycyrrhizin was capable of suppressing the LT-induced diarrhea at 10 mM. Because the exposure to glycyrrhizin compounds can induce hypermineralocorticoid-like effects in animal and human, these side effects may assuage the dehydration caused by diarrhea (Isbrucker and Burdock, 2006). In addition to glycyrrhizin, our previous study also demonstrated that oleanolic acid exhibits anti-diarrheal effects (Chen et al., 2007a). Therefore, these ndings suggested that oleananetype triterpenoids and glycyrrhizin might be considered as lead therapeutic agents for the treatment of ETEC-induced diarrhea. 5. Conclusions In this study, we evaluated the inhibitory abilities of TMH on the LTB and GM1 interaction by GM1 -ELISA. In silico analysis model (docking technique) was used to predict the effective phytochemicals from TMH extracts. The docking results indicated that triterpenoids, especially oleanane type, presented a better binding ability to LTB. Glycyrrhizin exhibited the lowest binding energy score among the oleanane-type triterpenoids. In vitro and in vivo models further showed that glycyrrhizin inhibited LT-induced diarrhea via the inhibition of GM1 and LTB interaction. Therefore, these ndings suggested that an LT inhibitor, glycyrrhizin, was found from TMH by in silico, in vitro, and in vivo analysis.

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Acknowledgments This work was supported by grants from National Research Program for Genomic Medicine, National Science and Technology Program for Agricultural Biotechnology, National Science Council, Committee on Chinese Medicine and Pharmacy, Department of Health (CCMP96-RD-201, CCMP97-RD-201), and China Medical University (CMU97-CMC-004 and CMU97-064), Taiwan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jep.2008.11.011. References
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