npentadactylon Larreat: Experimental and computational studies

Emiliano Tesoro-Cruz e, Samuela Instituto de Ciencias de la Salud, Universidad Autonomb Seccion de Estudios de Posgrado e Investigacion y Dep

11340, Mexico, D.F., Mexicoc Unidad de Investigacion Medica en Genetica Humanad Seccion de Estudios de Posgrado e Investigacion, Instie a, UMA

cion Sal

ga, UM

1988; Caceres, 1996; Chanfon, 2007). Chiranthodendron pentadactylon

e wetmalad for

mation, as well as an analgesic (Linares et al., 1988; Argueta et al.,

Contents lists available at SciVerse ScienceDirect

w.e

Journal of Ethno

Journal of Ethnopharmacology 143 (2012) 716719Velazquez et al., 2006; Calzada et al., 2006, 2010). Mostly thefercalber1@hotmail.com (F. Calzada).1994).Previous pharmacological investigations reported the antiproto-

zoal, vasorelaxant antibacterial, antipropulsive, and antisecretoryactivities of this plant (Perusqua et al., 1995; Alans et al., 2005;

0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.jep.2012.07.039

n Corresponding author. Tel.: 525 627 6900x21367; fax: 525 761 0952.E-mail addresses: fercalber10@gmail.com,monly known as or de manita and mano de leon in Mexico,while in Guatemala, it is locally called as manita (Linares et al.,

the treatment of several diseases such as heart illness, diarrhea,dysentery, epilepsy, cancer, ulcers, headache, eyes pain and inam-1. Introduction

Chiranthodendron pentadactylon Larreat (Sterculiaceae) is com-

is a small to medium sized evergreen tree which grows along thmixed oakpine and deciduous mountain forests from Guateand southern Mexico. In Mexican traditional medicine it is useVibrio cholerae toxin

Escherichia coli toxin

Antisecretory

Diarrhea

SDSPAGE analysis

Molecular docking

to conduct computational study.

Results: The antisecretory activity of epicatechin was tested against Vibrio cholera and Escherichia coli

toxins at oral dose 10 mg/kg in the rat model. It exhibited the most potent activity on Vibrio cholera

toxin (56.9% of inhibition). In the case of Escherichia coli toxin its effect was moderate (24.1% of

inhibition). SDSPAGE analysis revealed that both ()-epicatechin and Chiranthodendron pentadactylonextract interacted with the Vibrio cholera toxin at concentration from 80 mg/mL and 300 mg/mL,respectively. Computational molecular docking showed that epicatechin interacted with four amino

acid residues (Asn 103, Phe 31, Phe 223 and The 78) in the catalytic site of Vibrio cholera toxin, revealing

its potential binding mode at molecular level.

Conclusion: The results derived from computational, in vitro and in vivo experiments on Vibrio cholera

and Escherichia coli toxins conrm the potential of epicatechin as a new antisecretory compound and

give additional scientic support to anecdotal use of Chiranthodendron pentadactylon Larreat in Mexican

traditional medicine to treat gastrointestinal disorders such as diarrhea.

& 2012 Elsevier Ireland Ltd. All rights reserved.Unidad de Investigacion Medica en Inmunolog

Instituto Nacional de Ciencias Medicas y Nutrif Unidad de Investigacion Medica en Farmacolo

CP 06725 Mexico, D.F. Mexico

a r t i c l e i n f o

Article history:

Received 19 April 2012

Received in revised form

19 June 2012

Accepted 20 July 2012Available online 4 August 2012

Keywords:

(-)-Epicatechin

Flavan-3-ol

Chiranthodendron pentadactylon Larreat

Sterculiaceae-Basurto b, Normand Garcia-Hernandez c, Elizabeth Barbosa d,Calzada f, Fernando Calzada f,n

a del Estado de Hidalgo, Km. 4.5 Carretera Pachuca-Tulancingo, Unidad Universitaria, CP 42076 Pachuca, Hidalgo, Mexico

artamento de Bioqumica, Escuela Superior de Medicina, Instituto Politecnico Nacional, Plan de San Lus y Daz Miron, CP

, UMAE Hospital de Pediatra, Centro Medico Nacional Siglo XXI, IMSS, Mexico

tuto Politecnico Nacional, Escuela Superior de Medicina, Plan de San Lus y Daz Miron, CP 11340, Mexico, D.F., Mexico

E Hospital de Pediatra, Centro Medico Nacional Siglo XXI, IMSS, Departamento de Investigacion Experimental y Bioterio del

vador Zubiran, Mexico, D.F, Mexico

AE Hospital de Especialidades-21 Piso CORCE Centro Medico Nacional Siglo XXI, IMSS, Av. Cuauhtemoc 330, Col. Doctores,

a b s t r a c t

Ethnopharmacological relevance: Chiranthodendron pentadactylon Larreat is frequently used in Mexican

traditional medicine as well as in Guatemalan for several medicinal purposes, including their use in the

control of diarrhea.

Aim of the study: This work was undertaken to obtain additional information that support the

traditional use of Chiranthodendron pentadactylon Larreat, on pharmacological basis using the major

antisecretory isolated compound from computational, in vitro and in vivo experiments.

Materials and methods: ()-Epicatechin was isolated from ethyl acetate fraction of the plant crudeextract. In vivo toxin (Vibrio cholera or Escherichia coli)-induced intestinal secretion in rat jejunal loops

models and sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE) analysis on

Vibrio cholera toxin were used in experimental studies while the molecular docking technique was usedClaudia Velazquez a, Jose CorreaAnti-diarrheal activity of ()-Epicatechi

journal homepage: wwfrom Chiranthodendron

lsevier.com/locate/jep

pharmacology

pentadactylon Larreat, on pharmacological basis using epicatechin

column chromatography (CC) on a Sephadex LH-20 (25 g, Farmacia)

Technical specications for the production, care and use of

animals were sacriced and the intestinal loops were removed,

C. Velazquez et al. / Journal of Ethnopharmacology 143 (2012) 716719 717laboratory animals (SAGARPA, 2001).

2.5. Antisecretory assay

The antisecretory activity of methanol extract and ()-epica-techin was tested using a method previously describedby Velazquez et al. (2006, 2009). The antisecretory effect wasUV, IR, and [a]), MP, TLC and HPLC with authentic sample availablein our laboratory (Velazquez et al., 2009).

2.4. Animals

Male SpragueDawley rats (200250 g) were obtained fromthe animal house of the IMSS. The experimental protocols wereapproved by the Animal Care and Use Committee of PediatryHospital from Centro Medico Nacional Siglo XXI, IMSS. Investiga-tions using experimental animals were conducted in accordancewith the ofcial Mexican norm NOM 0062-ZOO-1999 titledusing CHCl3 in EtOH (9.5:0.5; 8:2; 7:3; 6:4; 5:5; 3:7; 1:9; v/v),MeOH (10) and water (10) to give eight fractions (F-1 to F-8).Fraction F-5 (300 mg) was resolved by high pressure liquid chro-matography (HPLC) with a Spherirorb S5ODS2 column (5% formicacidacetonitrile, 80:20, v/v ow rate of 3.2 mL min1, l 280 nm)allowing the isolation of ()-epicatechin (40 mg). Epicatechin wasidentied by comparison of the spectroscopic data (1H and 13C NMR,of Instituto Mexicano del Seguro Social (IMSS) where the voucherspecimen is conserved under reference number 14404.

2.2. Extraction from Chiranthodendron pentadactylon

The air-dried and nely powdered owers (10.0 kg) wereextracted by maceration at room temperature with MeOH(220 L). After ltration the extracts were combined and evapo-rated in vacuo to yield 1.2 kg of red residue.

2.3. Isolation of epicatechin from the MeOH extract of

Chiranthodendron pentadactylon

Epicatechin was isolated fromMeOH extract of Chiranthodendronpentadactylon according to the method of Velazquez et al. (2009).Briey, the MeOH extract (200 g) was suspended in 10% MeOHwater (350 mL) and successively partitioned with CH2Cl2 and EtOAc(350 mL3, 6.0 g). The EtOAc fraction (6 g), was subjected tofrom computational, in vitro and in vivo experiments.

2. Materials and methods

2.1. Plant material

The owers from Chiranthodendron pentadactylon (Sterculiaceae)were collected by Dr. Fernando Calzada in October 2001 inOzumba, State of Mexico, Mexico. The plant material was authen-ticated by MS Abigail Aguilar-Contreras of the Herbarium IMSSMphenolic compounds such as avonoids are responsible for theantisecretory effect of or de manita. Among them, epicatechinexhibited the most potent antisecretory activity (Velazquez et al.,2009). Thus, the present work was undertaken to obtain additionalinformation that support the traditional use of Chiranthodendronstudied on intestinal secretion indirectly by measuring the uidweighted and measures. The antisecretory activity of the sampleswas measured as the uid secretion in the loops and expressed inpercent of inhibition.

2.6. Sodium dodecyl sulphatepolyacrylamide gel electrophoresis

(SDSPAGE)

Vibrio cholerae or Escherichia coli toxin (10 mg) dissolved inwater was treated with epicatechin (0, 80 mg, 160 mg, 320 mg, and640 mg) or methanol extract (300 mg). After this, 3.4 mL of 2-mer-captoethanol was added and the mixture was kept for 10 min at40 1C. Then denaturated proteins were analyzed by SDSPAGEaccording to the Laemmli method (Laemmli, 1970) and stainedwith Coomassie-blue.

2.7. Computational study

To examine the interaction between epicatechin and choleratoxin, docking simulations were done on the 3-D structure oftoxin. Molecular docking simulations were performed usingversion 3.0.5 of the program AutoDock along with AutoDockToolsusing the hybrid Lamarckian Genetic Algorithm (LGA). Thisprogram was chosen because its algorithm allows full exibilityof small ligands. It has been shown that it successfully reproducesmany crystal structure complexes and includes an empiricalevaluation of the binding free energy. The preparation oftoxin and epicatechin input structure and the denition of thebinding sites were carried out under a GRID-based procedure.First, a rectangular grid box was constructed over toxin(126126126 A3). The setting up of the grids was performedwith 60 points in each dimension, with a spacing of 0.375 Abetween the grid points. In this study the X-ray structure ofcholera toxin was taken from the protein Data Bank. Previously,the toxin structure was cleaned of its water molecules. All dock-ing simulations were carried out with LGA and were realized withan initial population of 100 randomly placed individuals and amaximum number of energy evaluations (1107). Resultingdocking orientations within 1.0 A in the root-mean square devia-tion tolerance of each other were clustered together and repre-sented by the result with the most favorable free energy ofbinding.

2.8. Statistical analysis

The results are expressed as the mean7S.E.M, MannWhitneyU-test. Values with po0.05 were considered signicant.

3. Results and discussion

Diarrhea is a common symptom of gastrointestinal infectionscaused by a wide range of pathogens, including bacteria, virusesand protozoa. Among these Rotavirus, Escherichia coli, Shigella,Campylobacter, Salmonella, Vibrio cholerae, and Cryptosporidiumare responsible for most acute cases of diarrhea. In the case ofEscherichia and Vibrio the acute watery diarrhea occurs when theaccumulation in the intestine following Vibrio cholerae (Sigma) orEscherichia coli (heat-stable enterotoxins STa, Sigma) toxin admin-istration to rats. Two jejunal loops were prepared in the rats andinoculated with 3 mg/mL of toxin dissolved in 1 PBS with 1%bovine albumin. Rats (n11 per group by duplicate) were treatedorally with epicatechin (10 mg/kg in 1 mL of a 2% DMSO solution inwater), or vehicle (2% DMSO solution in water). Loperamide(10 mg/kg) was used as an antisecretory drug. After 4 h, thebalance between absorption and secretion in the small intestine is

disturbed by excessive secretion caused by bacterial enterotoxins(WHO, 2002; Casburn-Jones and Farthing, 2004; Khan et al., 2004;UNICEF/WHO, 2009). In Mexico, over the last 6 years the

gastrointestinal infections have been a serious health problem (SS,2008). Since 2006, a multidisciplinary approach has been developedby Calzada et al. (Velazquez et al., 2006, 2009) to evaluate theantisecretory potential of some Mexican medicinal plants and toisolate the active constituents that will directly inhibit intestinalsecretory mechanism and thereby reduce stool volume.

The results showed that epicatechin obtained from the owersof Chiranthodendron pentadactylon at oral doses of 10 mg/kg isactive against Vibrio cholera and Escherichia coli toxins withinhibition values of 56.1% and 24.0%, respectively (Table 1). Theinhibitory effect on Escherichia coli toxin conrms the potential ofepicatechin as a new antisecretory compound of therapeuticutility as an alternative treatment in the control of the diarrhea.Thus, it has a potential for the development of novel antidiarrhealdrugs. In addition, it validates the use of Chiranthodendronpentadactylon in Mexican traditional medicine in the treatmentof gastrointestinal disorders such as acute watery diarrhea(Velazquez et al., 2006, 2009).

Table 1In vivo antisecretory activity of ()-epicatechin isolated from Chiranthodendronpentadactylona.

Compound % inhibition against

Vibrio cholerae toxin Escherichia coli toxin

Epicatechin (10 mg/kg) 56.976.3 24715.6Loperamideb (10 mg/kg) 43.3713.2 nt

a The values represent the mean of the response in 11 rats 7SEM; two loopswere prepared in each animal and challenged with 3 mg of Vibrio cholerae orEscherichia coli toxins.

b Positive control; Po0.05.

Cholera toxin

A subunit

B subunit654321

Fig. 1. Binding properties of the epicatechin and MeOH extract of Chiranthodendronpentadactylon to cholera toxin analyzed by SDSPAGE. (1) Cholera toxin (CT) 10 mg(control). (2) CT: epicatechin; 10 mg: 80 mg. (3) CT: epicatechin; 10 mg: 160 mg.(4) CT: epicatechin; 10 mg: 320 mg. (5) CT: epicatechin; 10 mg: 640 mg. (6) CT: MeOHextract of Chiranthodendron pentadactylon; 10 mg: 300 mg.

O

OH

OH

OH

OH

OH

Fig. 2. Chemical structure of ()-epicatechin.

C. Velazquez et al. / Journal of Ethnopharmacology 143 (2012) 716719718Fig. 3. (Left) Structure of Vibrio cholerae toxin, A: subunit A and B: subunit B. (RigTo characterize the mode of action of the epicatechin oncholera toxin, we examined the interaction between differentconcentrations of the avan-3-ol and toxin using SDSPAGEanalysis (Fig. 1). The result revealed that both ()-epicatechin(Fig. 2) and Chiranthodendron pentadactylon extract interactedwith the Vibrio cholera toxin at concentration from 80 mg/mLand 300 mg/mL, respectively. In addition, when 8 mg of choleratoxin was treated with epicatechin of concentration 80640 mg/mL the band of the enzymatically active A subunit of the toxindecreased and disappeared, whereas the band of the B subunit ofthe toxin showed a weak inhibition. Similar effect was observedwhen the cholera toxin (8 mg) was treated with the Chiranthoden-dron pentadactylon crude extract (300 mg/mL). Results of the SDSPAGE analysis between cholera toxin and epicatechin can explainthe results previously obtained by Velazquez et al. (2009) withantisecretory avonoids from Chiranthodendron pentadactylon. Itis a direct evidence for a specic interaction of epicatechin withthe A subunit of cholera toxin.

In relation with the molecular docking experiments is impor-tant to note that they revealed that (Fig. 3) as ()-epicatechininteracts with four amino acid residues, which include Asn 103(at 3.10 A), Phe 31 (at 4.14 A), Phe 223 (at 3.28 A) and The 78 (at3.32 A). The results explain the mode of interaction of epicatechinwith the amino acid environment of the binding site in thecholera toxin. Also, in agreement with both docking and SDSPAGE analysis, they demonstrate that epicatechin is a stronginhibitor of cholera toxin and should be considered as a leadingcompound in the development of novel antisecretory agents inthe control of acute watery diarrhea.ht) Binding mode of epicatechin inside the active site of Vibrio cholera toxin.

Acknowledgment

The authors thank MS Abigail Aguilar, IMSS Herbarium, forauthentication of plant material.

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C. Velazquez et al. / Journal of Ethnopharmacology 143 (2012) 716719 719

Anti-diarrheal activity of (-)-Epicatechin from Chiranthodendron pentadactylon Larreat: Experimental and computational...IntroductionMaterials and methodsPlant materialExtraction from Chiranthodendron pentadactylonIsolation of epicatechin from the MeOH extract of Chiranthodendron pentadactylonAnimalsAntisecretory assaySodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)Computational studyStatistical analysis

Results and discussionAcknowledgmentReferences