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Journal of Ethnopharmacology 126 (2009) 455–458

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

ntisecretory activity from the flowers of Chiranthodendron pentadactylon and itsavonoids on intestinal fluid accumulation induced by Vibrio cholerae toxin in rats

laudia Velázqueza,∗, Fernando Calzadaa,∗, Baldomero Esquivelb, Elizabeth Barbosaa, Samuel Calzadaa

Unidad de Investigación Médica en Farmacología de Productos Naturales, Hospital de Pediatría, CORCE 2◦ piso Centro Médico Nacional Siglo XXI, IMSS, Av. Cuauhtémoc 330,ol. Doctores, CP 06725, México, D.F., MexicoInstituto de Química de la Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Delegación Coyoacán, CP 04510, México, D.F., Mexico

r t i c l e i n f o

rticle history:eceived 1 April 2009eceived in revised form 8 September 2009ccepted 10 September 2009vailable online 23 September 2009

eywords:hiranthodendron pentadactylon

a b s t r a c t

Ethnopharmacological relevance: The flowers of Chiranthodendron pentadactylon Larreat. (Sterculiaceae)has been traditionally used as folk medicine in Mexico for the treatment of gastrointestinal disorderssuch as diarrhea and dysentery.Aim of the study: This study aimed to assess the antisecretory activity which supports the therapeutic useof Chiranthodendron pentadactylon and its flavonoids to treat diarrhea.Materials and methods: The methanol extract of Chiranthodendron pentadactylon, subsequent fractions,and flavonoids were evaluated on cholera toxin-induced intestinal secretion in rat jejunal loops model.

terculiaceaelavonoidsntisecretory activityibrio cholerae toxin

Results: Three antisecretory flavonoids were isolated by bioassay-guided purification, namely, iso-quercitrin 3, (+)-catechin 4 and (−)-epicatechin 5. Among them, epicatechin exhibited the most potentantisecretory activity with ID50 of 8.3 �M/kg. Its potency was close that of to loperamide (ID50 6.1 �M/kg),drug used as control. Isoquercitrin (ID50 19.2 �M/kg) and catechin (ID50 51.7 �M/kg) showed moderateand weak activity, respectively.Conclusion: The results of the present study lend some support to the anecdotal report for the traditional

antho

use of the flowers of Chir

. Introduction

Secretory diarrhea occurs when the balance between absorp-ion and secretion in the small intestine is disturbed by excessiveecretion caused by bacterial enterotoxins. It is the leading cause ofeath in infants in developing countries and currently accounts forn estimated three million deaths each year among children underyears of age (Casburn-Jones and Farthing, 2004). In Mexico, lastyears the gastrointestinal infections have been a serious health

roblem and were the second cause of morbidity among all ageroups (SS, 2008).

A number of different bacteria can cause diarrhea by theroduction of highly potent enterotoxins, including: entero-oxigenic Escherichia coli, Salmonella typhi, Clostridium difficile,lostridium freudii, Aeromonas hydrophila, Yersinia enterocolitica,

ampylobacter jejuni and Vibrio cholerae. Specifically, heat-labilenterotoxin from Vibrio cholerae causes intestinal hypersecre-ion of fluid and electrolytes by activation of the cyclic AMPcyclic 3′,5′-adenosine monophosphate)-adenylate cyclase sys-

∗ Corresponding authors. Tel.: +525 627 6900x21367.E-mail addresses: fercalber1@hotmail.com (F. Calzada), cvg09@yahoo.com

C. Velázquez).

378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2009.09.016

dendron pentadactylon in the control of dysentery.© 2009 Elsevier Ireland Ltd. All rights reserved.

tem in the mucosal epithelium (Torregosa et al., 1996; Raufman,1998; Amstrong and Cohen, 1999; Casburn-Jones and Farthing,2004).

Oral rehydration therapy is regarded as the treatment of choiceto control diarrhea. It reduces the levels of mortality in childrenand adult by dehydration, but not morbidity for diarrhea. To treatthe secretory diarrhea there are some drugs, such as racecadotril,bismuth salicylate and loperamide that decrease intestinal hyper-secretion. However, these drugs have side effects; the first causebronchospasm, fever, and vomiting, the second cause tinnitus,black tongue and has a delayed onset of action. Loperamideshould not be administrated to patients with constipations, andintestinal obstruction, and in acute dysentery (Casburn-Jones andFarthing, 2004). Thus, the search for new antisecretory agents thatshould be effective and safe to treat diarrhea is still a necessarygoal.

Chiranthodendron pentadactylon Larreat. (Sterculiaceae), knownin Mexico as “flor de manita” and “macpaxochitl”, is a tree of12–15 m of height that grows in Mexico state and Guerrero; its

flowers have been used in Mexican traditional medicine sincethe Aztecs age to treat chronic ulcers, eye’s pain and inflam-mation; at present, it is used to treat heart illness, epilepsy,diarrhea and dysentery (Linares et al., 1988; Argueta et al.,1994; Lara and Márquez, 1996). The extracts have been reported

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56 C. Velázquez et al. / Journal of Eth

o exhibit diverse bioactivities such as anticholinergic, spas-olitic, antiprotozoal, antibacterial and antisecretory activities

Argueta et al., 1994; Perusquia et al., 1995; Alanís et al., 2005;elázquez et al., 2006; Calzada et al., 2006). Previous phytochem-

cal studies of this species revealed the presence of hydrocarbons,avonoids, steroids and sugars (Linares et al., 1988; Lara andárquez, 1996), but to the best of our knowledge, there have

een no prior studies on its antisecretory constituents. There-ore, the present work was undertaken to evaluate a possiblentisecretory activity of the crude extract, partially purifiedractions and flavonoids isolated from Chiranthodendron pen-adactylon.

. Materials and methods

.1. Plant material

The flowers from Chiranthodendron pentadactylon were pur-hased in the Sonora Market in Mexico City in October 2001 byalzada and Velázquez. The plant material was authenticated byS Abigail Aguilar-Contreras, of the Herbarium IMSSM of Institutoexicano del Seguro Social (IMSS) where the voucher specimen

No. 14256) was deposited.

.2. Extraction and fractionation

The air-dried flowers (10.26 kg) were ground and extracted byaceration at room temperature with MeOH (20 L× two times).fter filtration the solvent was evaporated in vacuo to yield.42 kg of red residue (13.84%). The MeOH active extract (409 g,7.1 ± 14.5 of inhibition at doses of 300 mg/kg) was suspended

n 10% MeOH–water (700 mL) and successively partitioned withH2Cl2 (700 mL × three times, 34.2 g, 28.2 ± 9.4% of inhibitiont doses of 50 mg/kg) and EtOAc (700 mL × three times, 15.2 g,8.2 ± 9.5 of inhibition at doses of 50 mg/kg). The aqueous residual

ayer (ARL, 186.2 g, 20.7 ± 9.0% of inhibition at doses of 50 mg/kg)as lyophilized.

.3. Isolation of flavonoids

The most active fraction (EtOAc, 15 g) was subject to columnhromatography (CC) on Sephadex LH-20 (100 g, Farmacia) usingHCl3 in EtOH (9.5:0.5; 8:2; 7:3; 6:4; 5:5; 3:7; 1:9; v/v), MeOH10) and water (10) to give eight fractions (F-1 to F-8). Fraction F-42.04 g) was purified by preparative TLC (silica gel 60 F254 Merck;tOAc–MeOH–water, 100:16.5:13.5) to give kaempferol-3-O-(6′ ′--E-p-coumaroyl)-�-d-glucopyranoside (tiliroside 1, 282.9 mg;P 209 ◦C), kaempferol-3-O-�-d-glucopyranoside (astragalin 2,

53.3 mg; MP 180 ◦C) and quercetin-3-O-�-d-glucopyranosideisoquercitrin 3, 173.8 mg; MP 188 ◦C). Fraction F-5 (731.1 mg) wasesolved by high pressure liquid chromatography (HPLC) with apherisorb S5ODS2 column (5% formic acid–acetonitrile, 80:20, v/vow rate of 3.2 mL min−1, � 280 nm) allowing the isolation of (+)-atechin (4, 245.7 mg; MP 240 ◦C) and (−)-epicatechin (5, 81.9 mg;P 182 ◦C). The isolated flavonoids were identified by compari-

on of the spectroscopic data (1H and 13C NMR, UV, IR, and [�]),LC and HPLC with authentic samples available in our laboratoryKuroyanagui et al., 1978; Lee et al., 1992; Liu et al., 1999; Calzadand Alanís, 2007).

.4. Animals

Male Sprague–Dawley rats (200–250 g) were obtained fromhe animal house of the IMSS. The experimental proto-ols were approved by the Animal Care and Use Commit-

rmacology 126 (2009) 455–458

tee of Pediatry Hospital from Centro Medico Nacional SigloXXI, IMSS. Investigations using experimental animals wereconducted in accordance with the official Mexican normNOM 0062-ZOO-1999 entitled Technical specifications for theproduction, care and use of laboratory animals (SAGARPA,2001).

2.5. Antisecretory assay

The antisecretory activity of methanol extract, fractions andpure compounds was tested using a method previously describedby Velázquez et al. (2006). The antisecretory effect was studiedon intestinal secretion indirectly by measuring the fluid accumula-tion in the intestine following Vibrio cholerae toxin administrationto rats. Two jejunal loops were prepared in the rats and inoc-ulated with 3 �g/mL of cholera toxin dissolved in 1× PBS with1% bovine albumin. Rats (n = 5 per group by duplicated) weretreated orally with the extract, fractions and pure compounds(300, 50 and 10 mg/kg in 1 mL of a 2% DMSO solution in water,respectively), or vehicle (2% DMSO solution in water). Loperamide(10 mg/kg) was used as antisecretory drug. After four hrs, the ani-mals were sacrificed using ethyl ether and the intestinal loops wereremoved, weighted and measures. The antisecretory activity of theextract, fractions and isolated compounds was measured as thefluid secretion in the loops and expressed in percent of inhibi-tion.

2.6. Acute toxicity study in rats

The acute toxicity of the MeOH extract was determined in maleand female Sprague–Dawley rats (body weight range 80–100 g) fol-lowing the methodology previously described by Lorke (1983). Theanimals were housed in a climate – and light – controlled roomwith a 12 h light/dark cycle. Twelve hours before experiments, foodwas withheld, but animals had free access to drinking water. Theanimals were randomly divided in five groups of three animalsper sex. Group 1 was the control vehicle (2% DMSO solution inwater), groups 2–5 were orally treated with extract at doses of 600,1200, 2400 and 4800 mg/kg. The extract was suspended in vehi-cle and was administered intragastrically in a volume not higherthan 10 mL/kg. The sample was administered at single doses. Thegeneral behavior of rats was observed daily in a period of 14 daysfor mortality, toxic effects and/or changes in behavioral pattern.At the end of the experiments the animals were sacrificed in aCO2 chamber. After, the animals were sacrificed and the inter-nal organs (stomach, gut, lungs, kidney, heart, spleen, and liver)were extracted and the gross pathological observations were per-formed.

2.7. Statistical analysis

The results are expressed as the mean ± S.E.M., Mann–WhitneyU-test. Values with p < 0.05 were considered significant.

3. Results and discussion

In the present study bioactivity-guided fractionation of theactive MeOH extract from the flowers of Chiranthodendron pen-tadactylon on cholera toxin-induced intestinal secretion in ratjejunal loops model was performed (Table 1). The MeOH extract

was divided into organic and soluble fractions by solvent partitionwith CH2Cl2 and EtOAc. All fractions (EtOAc, CH2Cl2 and ARL) weretested for antisecretory activity at doses of 50 mg/kg. As result ofthis process, the EtOAc-soluble fraction showed the best inhibitoryactivity (88.2% of inhibition). In order to isolate the active com-

C. Velázquez et al. / Journal of Ethnopha

Table 1In vivo antisecretory activity of MeOH extract, EtOAc fraction and pure compoundsisolated from Chiranthodendron pentadactylona.

Compound % Inhibition ID50 (�M/kg)

MeOH extract (300 mg/kg) 87.1 ± 14.5 –EtOAc fraction (50 mg/kg) 88.2 ± 9.5 –Tiliroside (1) – InactiveAstragalin (2) – InactiveIsoquercitrin (3) – 19.2Catechin (4) – 51.7Epicatechin (5) – 8.3Loperamideb – 6.1

a The values represent the mean of the response in five rats ±S.E.M., two loopsw

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ere prepared in each animal and challenged with 3 �g of cholera toxin.b Positive control; p < 0.05.

ounds in the EtOAc fraction, it was purified by CC, preparativeLC and HPLC. Three flavonol glycosides and two flavan-3-ols weresolated (Fig. 1), these components were identified as kaempferol--O-(6′ ′-O-E-p-coumaroyl)-�-d-glucopyranoside (tiliroside 1),aempferol-3-O-�-d-glucopyranoside (astragalin 2), quercetin--O-�-d-glucopyranoside (isoquercitrin 3), (+)-catechin 4, and−)-epicatechin 5 (Kuroyanagui et al., 1978; Lee et al., 1992;iu et al., 1999; Calzada and Alanís, 2007). The CH2Cl2 and ARLractions were discarded because they showed weak antisecretoryctivity.

The antisecretory activity of the individual flavonol gly-osides and flavan-3-ols isolated in the EtOAc fraction wereested on cholera toxin-induced intestinal secretion in rat jejunaloops model (Table 1). Among the isolated compounds (−)-picatechin 5 showed the best antisecretory activity on thentestinal secretion with an ID50 of 8.3 �M/kg, its antisecre-ory activity was close that of to loperamide (ID50 6.1 �M/kg),

rug used as positive control. Isoquercitrin 3 and (+)-catechinshowed moderate and weak antisecretory activity. Tiliroside

nd astragalin at doses tested were inactive. To our knowledge,his the first report of antisecretory of compounds 1–5. Also,his is the first bioassay-guided work to obtain the antisecretory

ig. 1. Chemical structures of isolated flavonoids from Chiranthodendron pentadactylon.

Compound R1 R2

1 H O-(6′ ′

2 H O-�-d3 OH O-�-d

4

5

rmacology 126 (2009) 455–458 457

compounds from the flowers of Chiranthodendron pentadacty-lon.

Flavonoids such as flavan-3-ols and flavonol glycosides havebeen considered as the active principles of many antidiarrheicplants. It has been speculated that are a consequence of theirinhibitory effects on protozoa and intestinal peristalsis as weredemonstrated by in vitro and in vivo test. Isoquercitrin isolatedfrom Psidium guajava showed a spasmolytic effect of guinea-pig ileum (Morales et al., 1994). Tiliroside and (−)-epicatechinobtained from Helianthemum glomeratum and Rubus coriifolius,respectively, showed antiprotozoal activity against Entamoeba his-tolytica and Giardia lamblia (Alanís et al., 2003; Calzada et al., 2006;Barbosa et al., 2007; Calzada and Alanís, 2007). Data obtainedin this investigation suggest that (−)-epicatechin, isoquercitrinand tiliroside may play an important role in antidiarrhoeal prop-erties of Chiranthodendron pentadactylon in Mexican traditionalmedicine. Also, the results obtained in this work are in agree-ment and can explain the result previously obtained by Hör etal. (1995) with antisecretory oligomeric proanthocyanidins fromGuazuma ulmifolia which monomeric unit are (+)-catechin and (−)-epicatechin.

On the other hand, although the data are limited, thestructure–effect correlations revealed that antisecretory activityseems to be related to the non-planarity of the A and C ring ofthe flavan-3-ol structure. Moreover, the 2,3-cis (�) stereochem-istry might be an important requirement for high antisecretoryactivity since compound 4 which is trans-configurated is lessactive as antisecretory agent than cis-compound 5. In summary,the result of the present study along with the properties pre-viously described of (−)-epicatechin suggest that it may be aleading compound in the development of novel antidiarrheicagents.

From this study, it was observed that methanol extract didnot cause toxicity signs and death to a dose of 4800 mg/kg

p.o.

Finally, this work provides scientific evidence to support theanecdotal report for the traditional use of the flowers of Chiranth-odendron pentadactylon in the treatment of dysentery in Mexicantraditional medicine (Linares et al., 1988).

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58 C. Velázquez et al. / Journal of Eth

cknowledgments

The authors thank MS Abigail Aguilar, IMSS herbarium, foruthentication of plant material, 1H and 13C NMR Hector Rios, Ma.e los Ángeles Pena, IR Rocio Patino, MS Luis Velasco and Francisco

avier Pérez.

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