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  Journal of Ethnopharmacology 121 (2009) 372–378

Contents lists available at ScienceDirect

 Journal of Ethnopharmacology

  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 / j e t h p h a r m

Identification 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, Taiwanb Molecular Biology Laboratory, Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwanc Department of Biochemistry, China Medical University, Taichung, Taiwand Department of Microbiology, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan

a r t i c l e i n f o

 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

a b s t r a c t

Ethnopharmacological relevance: Glycyrrhiza uralensis has been used for the treatment of gastrointestinal

disorders,suchas diarrhea,in severalancientcultures. Glycyrrhizinis theprincipalcomponentof 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 conse-

quently leading to the massive loss of fluid 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 diar-

rhea, 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 othertypes of triterpenoids. Moreover, by in vitro and in vivo models, we demonstrated that glycyrrhizin was

the most effective oleanane-type triterpenoid that significantly suppressed both the LTB-binding ability

(IC50 =3.26±0.17 mM) and the LT-induced fluid 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.

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 traveler’s

  Abbreviations: E. coli, Escherichiacoli; ETEC,Enterotoxigenic Escherichiacoli; GM1-

ELISA, GM1-enzyme-linkedimmunosorbent assay;LT,heat-labile enterotoxin; LTA,A

subunitA ofLT;LTB,B subunitof 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.

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 sub-

unit and five 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 [Gal1-3GalNAc1-4

(Neu5Ac2-3)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, fol-

lowed by the translocation of A subunit (LTA) into the cells. Inside

the intestinal cells, LTA catalyzes the ADP-ribosylation of the stimu-

latory GTP-binding protein, resulting in the increased intracellular

levels of cyclic AMP. Elevated levels of cyclic AMP in the cells cause

massive loss of fluid andionsfromthe cells,consequentlyleading to

the symptom of diarrhea (Spangler, 1992). Therefore, the binding

of LTB to GM1 is an attractive target for developing drugs or pro-

0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.jep.2008.11.011

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 Table 1

The inhibitory abilities of TMH, which have ethnomedically been used for the treatment of diarrhea.

Herbal name Latin name Family Inhibitory ability (%)

Plumula Nelumbinis Nelumbo nucifera Nymphaeaceae 79.3

Cortex Cinnamomi Cinnamomon cassia Lauraceae 76.3

Rhizome Coptis Coptis chinensis Ranunculaceae 73.7

Radix Glycyrrhizae Glycyrrhiza uralensis Leguminosae 73.3

Flos Caryophylli Eugenia caryophyllata Myrtaceae 72.9

Cortex Magnoliae Officinalis Magnoliae officinalis Magnoliaceae 71.8

Radix Angelicae Sinensis Angelica sinensis Umbelliferae 71.4Radix Bupleuri Bupleurum chinense Umbelliferae 71.1

Rhizoma Alismatis Alisma orientalis Alismataceae 69.5

Radix Ginseng Panax ginseng  Araliaceae 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 officinale (Chen et al., 2007b), the leaf of  Camellia japon-

ica (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 millen-

nia. Therefore, we collected TMH, which have been ethnomedically

used for gastrointestinal disorders, in this study. By G M1-enzyme-

linked 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 inhi-

bition of GM1 and LTB interaction. These findings suggested that

glycyrrhizin might be a potent candidate for the treatment of LT-

induced 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 qual-

ities of TMH samples were verified by authors (Prof. Yuan-Shiun

Chang and Dr. Jaw-Chyun Chen) according to the Pharmacopoeia

Commission of People’s Republic of China (2005). The voucher

specimens were deposited in the Molecular Biology Laboratory,

Graduate Institute of Chinese Medical Science, China Medical Uni-

versity. Specimen number was listed in supplemental table. TMH

sample was ground with the homogenizer to a fine powder and

extracted by mixing 100g of herb powder with methanol at 4 ◦C

overnight. The supernatant was then collectedand 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 purification of Escherichia coli (E. coli) LT and

LTB

Recombinant LT and LTB were expressed in E. coli

BL21(DE3)pLysS strain and purified by affinity chromatogra-

phy as described previously (Chen et al., 2006). Briefly, 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 Tris–HCl, pH 7.5, 1 mM EDTA, 200 mM NaCl,

3mM NaN3), lysed by sonication, and centrifuged at 15,000× g 

for 20min 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 sulfate-

polyacrylamide gel electrophoresis and quantified 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 (16ng) was mixed with various amounts of com-pounds andincubated at 4 ◦C for 3 h with shaking. Microtiter plates

(MaxiSorp Nunc-ImmumTM plates, Nunc, Denmark) were coated

with 100l of 2ng/l GM1 (Sigma, St. Louis, MO, USA), which was

diluted in phosphate-buffered saline (PBS) (137 mM NaCl, 1.4 mM

KH2PO4, 4.3mM Na2HPO4, 2.7 mM KCl, pH 7.2), per well and incu-

bated 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 1h,

andthen incubatedwith100l of biotinylatedLTB/compound mix-

ture at 37 ◦C for 1 h. After three washes with washing buffer, 50l

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. Inhibitoryabilities of TMHon theinteractionbetween LTBand GM1. Numbers

inthebracketsarethesumofherbspeciesinthefamily. * p <0.05, ** p < 0.01, compared

with LTB.

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374 J.-C. Chen et al. / Journal of Ethnopharmacology 121 (2009) 372–378

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, 50l of chromogenic substrate, 2,2-azinobis(3-

ethylbenzthiazoline-sulfonic acid) (Sigma, St. Louis, MO, USA),

was added to each well and incubated at 37 ◦C for 15min. The

absorbance was read at 405nm 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 pre-

viously (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 file was in the PDBQ format, which

is an extension of the PDB format. The PDBQ format of lig-

ands (phytochemicals) was generated by Dundee’s PRODRG

server (http://davapc1.bioch.dundee.ac.uk/programs/prodrg/)

(Schüttelkopf and van Aalten, 2004). The PDB file (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 file 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, Tai-wan). Mouse experiments were conducted under ethics approval

fromthe ChinaMedical 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). Briefly, five mice per group were starved

with water only for 16 h. Each mouse was inoculated intragastri-

cally with 0.5 ml of 10g LT alone or in conjunction with various

amounts of TMH extracts or compounds. Six hours latter, mice

were sacrificed. The entire intestine from duodenum to rectum

was carefully removed to retain any accumulated fluid, 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

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 Table 2

Binding energy scores of oleanane type of triterpenoids.

Name Structure 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. Student’s t -test was used

for a comparison between two experiments. A value of  p < 0.05 was

considered statistically significant.

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Fig. 3. Inhibitory ability of glycyrrhizinon thebinding of LTB to GM1 by competitive

GM1-ELISA. Values are mean±S.E. of triplicate assays. ** p < 0.01, compared with LTB

treatment.

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 bind-ing 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, exhibitedapproximately60%

inhibitions at 1g/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, flavonoids, 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 phytochem-

icals 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 triter-

penoids presented better binding abilities than other triterpenoids,

and its average binding energy score was −11.14±2.21kcal/mol

(Fig. 2b). The docking data from part of oleanane-type triter-

penoids 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. Gly-

cyrrhizin significantly blocked the binding of LTB to G M1, with

the IC50 value of 3.26±0.17 mM (Fig. 3). It also significantly sup-

pressed the LT-induced fluid accumulation at 10 mM (Fig. 4).

Therefore, these findings indicated that the glycyrrhizin was

the likely active component for the suppression of LT-induced

diarrhea.

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.

 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 fitted LTB very well, with the pre-

dicted binding energy score of −18.03 kcal/mol. Hydrogen bonds

were formed directly between the carboxylic ketone group of gly-

cyrrhizin and Asn

90

residue of LTB. Extra hydrogen bonds were

Fig. 5. Docking structure between LT and glycyrrhizin. (A) Surface representation

of LTB complexes with glycyrrhizin. (B) Close up view of LTB complexes with gly-

cyrrhizin. Glycyrrhizin is represented by stick style. Side chains of selected residues

arerepresented byred wirestyle.Hydrogen bonds betweenLTB andglycyrrhizinare

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 resis-

tance, particularly in endemic areas (Garget al.,2000). Antimotility

agents like loperamide can lessen stool frequency and volume in

mild diarrhea. However,such agents are notsuitable in severe diar-

rhea because they may cause pooling of large amounts of fluid

in paralyzed bowel loops (Field, 2003). Additionally, when lop-

eramide 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 out-

weigh benefits (Li et al., 2007). The most commonly principle of 

management for serious diarrhea is the fluid replacement com-

bined with pharmacologic therapy (Ahlquist and Camilleri, 2001).Fluid and electrolyte replacement is used to replace fluid losses

by the administration of sugar-electrolyte solutions; however, it

does not facilitate the re-adsorption of secreted fluid and there-

fore does not lessen diarrhea. Because there is currently no specific

prophylaxis against diarrhea caused by bacterial toxin, the discov-

ery of agents that can block the function of toxin is important

for the prevention and treatment of bacterial toxin-induced diar-

rhea.

To develop the agents that inhibit the toxin-induced diarrhea

with specificity,we setup theLT- instead of castoroil-induced diar-

rheal model. Castor oil is experimentally used for the induction of 

diarrhea in animalmodel(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 liber-ation of ricinoleic acid from castor oil, results in the irritation and

inflammation 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

fluid accumulation assayand patent mouse gut assayare 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 abun-

dant 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 exam-ples, 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 suc-

cessfully and rapidly integrated rational approaches that exploit

and evolve the structural diversity provided by nature (Haustedt et

al.,2006). Therefore, in silicoscreeninghas shown a greatpromisein

drug discoverybecauseit playsan importantrolein digging outlead

(active) compounds from traditional medicine (Shen et al., 2003).

In this study, we setup a 3D structure library of 600phytochemicals

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 triter-

penoids in these families, except Convolvulaceae and Solanaceae

(Vincken et al., 2007). Oleanane-type triterpenoids are widely dis-

tributed 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 demon-

strated that three kinds of triterpenoids, including oleananic acid,

ursolic acid and betulinic acid, dock into the active site of LTB

(Chen etal., 2007a). Theirbinding inhibitory abilitiesare concentra-

tion dependent, with the IC50 value of 202.8±47.8, 493.6±100.0,

and 480.5±56.9M, respectively. These results indicated that

oleananic acid (oleanane type) was more active than ursolic acid

(ursane type) and betulinic acid (lupane type) in the suppres-

sion of LTB and GM1 interaction. Therefore, we suggested thatoleanane-type triterpenoids presented better abilities than other

types of triterpenoids in the inhibition of LTB and GM1 interac-

tion.

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, gas-

trointestinal, 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 hep-atoprotective effects. In this study, we first demonstrated that

Glycyrrhiza uralensis extract and glycyrrhizin reduced the bind-

ing of LTB to GM1, with the inhibitory abilities of 73.3 and

97.5%,respectively. Moreover, glycyrrhizinwas capable of suppress-

ing 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 demon-

strated that oleanolic acid exhibits anti-diarrheal effects (Chen

et al., 2007a). Therefore, these findings suggested that oleanane-

type triterpenoids and glycyrrhizin might be considered as lead

therapeutic agents for the treatment of ETEC-induced diar-

rhea.

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 phy-

tochemicals from TMH extracts. The docking results indicated that

triterpenoids, especially oleanane type, presented a better bind-

ing 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 diar-

rhea via the inhibition of GM1 and LTB interaction. Therefore, these

findings suggestedthat an LTinhibitor,glycyrrhizin, wasfound 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 Coun-

cil, 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.

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