Journal of Ethnopharmacology 103 (2006) 66–70

Antisecretory activity of plants used to treat gastrointestinaldisorders in Mexico�

Claudia Velazqueza,d,∗, Fernando Calzadaa,∗, Javier Torresb,Felipe Gonzalezc, Guillermo Ceballosd

a Unidad de Investigacion Medica en Farmacologıa de Productos Naturales, Hospital de Pediatrıa, 2◦ Piso, Centro Medico Nacional Siglo XXI,IMSS, Av. Cuauhtemoc 330, Col. Doctores, CP 06725, Mexico D.F., Mexico

b Unidad de Investigacion Medica en Enfermedades Infecciosas y Parasitarias, Hospital de Pediatrıa, 2◦ Piso, Centro Medico Nacional Siglo XXI,IMSS, Av. Cuauhtemoc 330, Col. Doctores, CP 06725, Mexico D.F., Mexico

c Unidad de Investigacion Medica en Infectologıa e Inmunologıa, Hospital de Infectologıa, 2◦ Piso, Centro Medico Nacional La Raza,IMSS, Av. Jacarandas, S/N, CP 02990, Mexico D.F., Mexico

d Instituto Politecnico Nacional, Escuela Superior de Medicina, Plan de San Luis y Dıaz Miron, CP 11340, Mexico D.F., Mexico

Received 9 August 2004; received in revised form 24 June 2005; accepted 29 June 2005Available online 19 September 2005

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Aqueous and methanolic extracts from 26 medicinal plants used in Mexico to treat gastrointestinal disorders were screened to entisecretory activity on cholera toxin-induced intestinal secretion in rat jejunal loops model. Extracts were tested at a dose of 3rom 56 samples tested, both extracts fromChiranthodendron pentadactylon, Hippocratea excelsa andOcimum basilicum were the mosotent with inhibition values ranging from 68.0 to 87.6%. On the other hand, the methanolic extract ofGeranium mexicanum (aerial partsnd the aqueous extract ofBocconia frutescens showed the highest activity with inhibition values of 93.4 and 86.0%, respectively. Thebtained in this study give some scientific support to the use of the Mexican medicinal plants employed for the treatment of gastrisorders such as diarrhea.2005 Elsevier Ireland Ltd. All rights reserved.

eywords: Mexican medicinal plants; Aqueous extracts; Methanolic extracts; Antisecretory activity; Cholera toxin

. Introduction

Diarrheal disease is one of the most common causesf morbidity and mortality in many developing countriesGuerrant, 1985; Amstrong and Cohen, 1999). It is oftenaused by enterotoxins which are produced by bacteriauch as enterotoxigenicEscherichia coli, Salmonella typhi,almonella typhimurium, Clostridium difficile, Clostrid-um freundii, Aeromonas hydrophila, Yersinia enterocolitica,ampylobacter jejuni andVibrio cholerae. Enterotoxins have

heir effect on the enterocyte functions by stimulating the

� Part of this work was taken from the Ph.D. thesis of Claudia Velazquez.∗ Corresponding authors. Tel.: +525 627 6900x22410; fax: +525 761 0952.

E-mail address: fercalber1@hotmail.com (F. Calzada).

secretion of transepithelial electrolytes, thus increasingosmotic flux of water and ions to the intestinal lumen. Speically, heat-labile (LT) and heat-stable (ST) enterotofrom Escherichia coli, Vibrio cholerae andCampylobacterjejuni increase net fluid secretion by affecting the enzyadenylyl cyclase or guanylyl cyclase in the intestinal mu(Guerrant, 1985; Torres et al., 1993; Mutschler et al., 1Hor et al., 1995; Torregosa et al., 1996; Raufman, 1Amstrong and Cohen, 1999).

To control diarrhea, the treatment of choice is oral redration solution (ORS). It has reduced the levels of mortin children and elderly by dehydration, but not morbiditydiarrhea (Amstrong and Cohen, 1999; Turvill et al., 200).To treat the secretory diarrhea there are some drugs, suracecadotril and loperamide, that decrease intestinal h

378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2005.06.046

C. Velazquez et al. / Journal of Ethnopharmacology 103 (2006) 66–70 67

secretion. Both drugs have side effects; racecadotril causebrochospasm, fever and vomiting, and loperamide should notbe administrated to children younger than 6 years of age andpatients with constipation, and intestinal obstruction, and inacute dysentery caused by bacterial infections because it canmask the symptoms and cause intestinal perforation (Brown,1979; Roge et al., 1993; Salazar et al., 2000).

On the other hand, there are some compounds that showedinhibitory properties on the intestinal secretion, such berber-ine, chlorpromazine, clonidine, nicotinic acid, indometacin,somatostatin and ethycrynic acid, but they were not devel-oped as antidiarrheal drugs (Rabbani et al., 1982; Sack andFroehlich, 1982; Fedorak and Field, 1987).

In Mexico, the use of medicinal plants to treat gas-trointestinal disorders such as diarrhea and dysentery iswidespread (Campos, 1991; Aguilar et al., 1994). However,most of these plant species have not been investigated froma pharmacological point of view to demonstrate their antise-cretory properties, which could lead to support their use asantidiarrheal and antidysenteric drugs in traditional medicine.

In this work, we screened 56 aqueous and methanolicextracts from 26 Mexican medicinal plants to assess theirantisecretory activity using the cholera toxin-induced intesti-nal secretion in rat jejunal loops model. It is important tonotice that none of these species or their isolated metaboliteshave been previously evaluated as antisecretory agents.

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pended in 1 mL of sterile water. For the study, aliquots of thetoxin solution were dissolved in a 1× PBS (NaCl 8 g, KCl0.2 g, Na2HPO4·7H2O 0.115 g, KH2PO4 0.2 g/L) solutionwith 1% bovine serum albumin (Sigma) to obtain a concen-tration of 3�g/mL.

2.4. Antisecretory assay

The antisecretory activity of the extracts was tested usinga method previously described (Torres et al., 1993). MaleSprague–Dawley rats (200–250 g) were obtained from theanimal house of the IMSS. The experimental protocols wereapproved by the Animal Care and Use Committee of Hospitalde Pediatria del Centro Medico Nacional Siglo XXI, IMSS, inaccordance with the guidelines for care and use of laboratoryanimals. The effect of the extracts was studied on intestinalsecretion indirectly by measuring the fluid accumulation inthe intestine following cholera toxin administration to rats.Two jejunal loops were prepared in the rats and inoculatedwith 3�g/mL of cholera toxin dissolved in 1× PBS with 1%bovine albumin. Rats (n = 4 per group by duplicated) weretreated orally with each extract (300 mg/kg in 1 mL of a 2%DMSO solution in water), or vehicle (2% DMSO solutionin water). Loperamide (10 mg/kg) was used as antidiarrhealdrug, After 4 h, the animals were sacrificed using ethyl ether.The antisecretory activity of the extracts was measured ast ntageo

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. Materials and methods

.1. Plant materials

The assessed plants were collected by the authors (Cand Velazquez) from different regions in Mexico: Mexiity, States of Hidalgo, Mexico, Sinaloa, Guanajuatoucatan. They were selected according to their use in M

can traditional medicine to treat gastrointestinal disordoucher herbarium specimens (Table 1) have been authenated by MS Abigail Aguilar, Herbarium IMSSM of Instituexicano del Seguro Social. Plant species, part used, vo

pecimens and the percentage of inhibition of the intesecretion are shown inTable 1.

.2. Preparation of crude extracts

The air-dried plant material (20 g) was extracted by mration with 300 mL of MeOH for 1 week. Then the macte was filtered and concentrated under reduced press0◦C. For aqueous extracts, 20 g of air-dried plant matas extracted by decoction with 100 mL of distilled water0 min, the solution was filtered and lyophilized. The yiere shown inTable 1.

.3. Cholera toxin

Lyophilized powder (1 mg) of cholera toxin (Sigma) coaining approximately 220,000 units/mg of protein was

t

he fluid secretion in the loops and expressed in percef inhibition.

.5. Statistical analysis

Values are expressed as mean± S.E.M. Statistical signifcance was determined using Mann–WhitneyU-test. Valueith p < 0.05 were considered significant.

. Results and discussion

We tested 56 aqueous and methanolic crude extbtained from medicinal plants used in Mexican traditioedicine for the treatment of gastrointestinal disordhese were tested at oral doses of 300 mg/kg becauseose most of the extracts showed antisecretory activit

raditional medicine since infusions or decoctions are usaken three times per day when diarrhea occurs, our rean be related with their traditional use because the useds approximately one cup of plant tea which is recommeny Mexican people to treat gastrointestinal disorders (Aguilart al., 1994).

The antisecretory activity was tested using the chooxin-induced intestinal secretion in rat jejunal loops mohe antisecretory activity of the extracts tested is showable 1. We found that both extracts fromChiranthodendronentadactylon, Hippocratea excelsa andOcimum basilicumere the most active with inhibition values ranging from 6

o 87.6% at 300 mg/kg. Methanolic extract ofGeranium mex-

68 C. Velazquez et al. / Journal of Ethnopharmacology 103 (2006) 66–70

Table 1Antisecretory activity of methanolic and aqueous extracts of selected Mexican medicinal plants at 300 mg/kg on intestinal secretion response to cholera toxin

Family Plant specie Part used Voucher number Extract % (w/w) yield % Inhibition

Liliaceae Allium sativum L. EB 14396 Methanolic 1.2 18.4± 10.8Aqueous 10.7 14.9± 5.5

Verbenaceae Aloysia triphylla (L’H er) Britton AP 126110 Methanolic 7.1 7.8± 4.7Aqueous 17.4 80.4± 22.8

Annonaceae Annona cherimola Miller S 14401 Methanolic 8.7 79.5± 14.6Aqueous 3.4 50.2± 12.9

Asteraceae Artemisa absinthium L. AP 12609 Methanolic 10.8 9.1± 2.3Aqueous 20.7 20.7± 11.1

Asteraceae Artemisa ludoviciana Nutt AP 14409 Methanolic 12.7 5.7± 2.9Aqueous 15.9 15.0± 6.8

Papaveraceae Bocconia frutescens L. AP 12618 Methanolic 9.7 24.1± 15.4Aqueous 14.3 86.0± 9.8

Leguminosae Caesalpinia pulcherrima (L.) Sw. AP 13591 Methanolic 23.0 16.3± 6.6Aqueous 23.8 12.8± 3.8

Caricaceae Carica papaya L. S 14397 Methanolic 6.7 20.9± 5.9Aqueous 23.3 13.6± 11.7

Chenopodiaceae Chenopodium ambrosioides L., green variety AP 14402 Methanolic 10.7 43.4± 6.5Aqueous 13.8 48.7± 11.6

Chenopodiaceae Chenopodium ambrosioides L., red variety AP 14395 Methanolic 13.9 19.7± 7.5Aqueous 21.1 16.7± 6.7

Chenopodiaceae Chenopodium murale L. AP 13592 Methanolic 8.3 31.9± 5Aqueous 15.9 32.8± 9.9

Sterculiaceae Chiranthodendron pentadactylon Larreat F 14104 Methanolic 17.7 87.6± 15.3Aqueous 12.6 84.8± 17.4

Asteraceae Chrysactinia mexicana A. Gray AP 14407 Methanolic 8.4 0Aqueous 23.1 0

Palmae Cocos nucifera L. EF 14398 Methanolic 29.1 23.4± 11.9Aqueous 19.4 12.5± 5.4

Convolvulaceae Dichondra argentea Humb & Bonpl AP 0103m Methanolic 12.1 22.3± 13.2Aqueous 16.5 36.7± 12.1

Moraceae Dorstenia contrajerva L. AP 14406 Methanolic 8.6 24.4± 16.4Aqueous 0.3 44.8± 5.9

Geraniaceae Geranium mexicanum H. B. & K. AP 14405 Methanolic 15.5 93.4± 6.7Aqueous 31.0 42.1± 15.2

Geraniaceae Geranium mexicanum H. B. & K. R 14405 Methanolic 14.6 0Aqueous 15.6 0

Hippocrateaceae Hippocratea excelsa H. B. & K. R 14394 Methanolic 7.2 80.3± 21.3Aqueous 15.5 75.0± 24.9

Verbenaceae Lippia alba (Mill.) N.E.Br. AP 14146 Methanolic 8.5 32.0± 9.8Aqueous 20.4 26.0± 9.1

Schizaeaceae Lygodium venustum Sw. AP 13270 Methanolic 11.2 51.6± 15.6Aqueous 12.3 0

Compositae Matricaria recutita L. AP 14399 Methanolic 19.9 25.4± 7.6Aqueous 30.5 24.3± 3.6

Labiatae Ocimum basilicum L. AP 14393 Methanolic 10.5 68.7± 19.7Aqueous 23.1 68.0± 20.8

Punicaceae Punica granatum L. EF 14403 Methanolic 48.2 55.9± 3.6Aqueous 28.5 19.1± 6.9

C. Velazquez et al. / Journal of Ethnopharmacology 103 (2006) 66–70 69

Table 1 (Continued )

Family Plant specie Part used Voucher number Extract % (w/w) yield % Inhibition

Rutaceae Ruta chalepensis L. AP 14400 Methanolic 23.0 73.7± 7.01Aqueous 23.9 23.6± 9.27

Anacardiaceae Schinus molle L. AP 14408 Methanolic 18.4 0Aqueous 12.8 79.5± 17.7

Leguminosae Senna villosa Mills L 14410 Methanolic 9.4 11.9± 3.4Aqueous 35.7 29.3± 17.9

Labiatae Thymus vulgaris L. AP 13594 Methanolic 9.4 0Aqueous 12.0 18.9± 10.3

Loperamide – – – – – 43.3± 13.1

The antisecretory activity was expressed as the percentage inhibition (mean± S.D.,n = 8) compared to the blank. Loperamide was used as a positive control(10 mg/kg). AP, aerial parts; EB, bulb epidermis; EF, fruit exocarp; F, flowers; L, leaves; R, roots; S, seed.p < 0.05 is considered to be significant.

icanum (aerial parts) and the aqueous extract ofBocconiafrutescens were the most active with inhibition values of 93.4and 86.0%, respectively.

On the other hand, the methanolic extract ofChenopodiumambrosioides green variety (aerial parts),Lygodium venus-tum, Punica granatum and Ruta chalepensis, the aqueousextracts ofAloysia triphylla, Chenopodium ambrosioidesgreen variety (aerial parts),Dorstenia contrajerva andSchi-nus molle showed inhibitory activity with values rang-ing from 43.4 to 79.5%. The 87% of the extracts testedshowed inhibitory activity of the intestinal secretion; onlyseven extracts did not show activity. In general, among theresearched extracts, the methanolic extracts exhibited thehighest antisecretory activity.

4. Conclusion

Some of the medicinal plants tested showed interest-ing antidiarrheal activity in vivo model. Both extracts ofAnnona cherimola, Chiranthodendron pentadactylon, Hip-pocratea excelsa, Ocimum basilicum, Geranium mexicanumgreen variety (aerial parts), methanolic extracts fromRutachalepensis, Lygodium venustum, Punica granatum, aqueousextracts ofBocconia frutescens, Aloysia triphylla, Dorsteniacontrajerva andSchinus molle, showed better antisecretorya oset com-p ssay-g inci-p

p ds,s owedmbbnl gara et

frutescens alkaloids have been isolated and the methano-lic and chlorophorm extracts showed antibacterial activityagainstVibrio cholera, a causal agent of diarrhea (Perez,2000; Caballero et al., 2002, 2003). The antiprotozoal orantibacterial activity seen in the most active extracts of thisstudy together with the antisecretory effect are evidence thatsupport the use ofChiranthodendron pentadactylon, Gera-nium mexicanum and Bocconia frutescens in Mexican tra-ditional medicine to treat diarrhea. It is important to noticethat no extract or isolated compound of these species hasbeen previously evaluated to demonstrate their antisecretoryactivity.

Some studies have been performed in order to find anti-secretory compounds from plants used in the traditionalmedicine to treat various kinds of diarrheas, in this sensethe extracts ofCroton urucurana, Croton lechleri, Berberisaristata and Guazuma ulmifolia were studied against theintestinal secretion caused byVibrio cholerae toxin. In thecases ofBerberis aristata, Guazuma ulmifolia and Cro-ton lechleri their antisecretory compounds were berberineand oligomeric proantocyanidins, respectively. FromCro-ton urucurana saponins, steroids, alkaloids, antocianidinsand catechins have been isolated. InGuazuma ulmifolia(Hor et al., 1995) and Croton urucurana, proanthocyani-dins and catechins can probably be associated with theirantisecretory activity (Dutta and Panse, 1962; Sack andF ;G al.,2

eda se ofC rald tsf to-d

inalp singt ed int ctedm t ofg

ctivity than loperamide. These results allows to prophese species as a potential sources of antisecretoryounds and should be therefore subjected to further bioauided phytochemicals studies to obtain their active prles.

From the most active species,Chiranthodendronentadactylon, several compounds including flavonoiteroids, hydrocarbons, sugars and gallic acid, which shoderate activity against the protozoaGiardia lamblia, haveeen isolated (Lara and Marquez, 1996; Alanis et al., 2003). Aioassay-guided study on the antiprotozoal activity ofGera-ium mexicanum againstEntamoeba histolytica andGiardiaamblia led to the isolation of a steroid, an alkaloid, a sund two flavan-3-ols, and (−) epicatechin was found to b

he active compound (Calzada et al., 2005). FromBocconia

roehlich, 1982; Fedorak and Field, 1987; Hor et al., 1995abriel et al., 1999; Gurgel et al., 2001; Fischer et004).

It is important to notice that our active extracts showntisecretory activity to 300 mg/kg whereas that in the caroton urucurana it showed antisecretory activity at an oose of 600 mg/kg (Gurgel et al., 2001). The active extrac

ound in this work will be an option to develop novel phyrugs useful to treat fluid loss in diarrhea.

The antisecretory compounds isolated from mediclants combined with ORS might be useful in decrea

he mortality caused by dehydration. The results obtainhis study give some scientific support to the use of seleedicinal plants in Mexican tradition for the treatmenastrointestinal disorders such as diarrhea.

70 C. Velazquez et al. / Journal of Ethnopharmacology 103 (2006) 66–70

Acknowledgements

MS Abigail Aguilar, IMSS herbarium, for authenticationof plant material and Antonio Cervantes. This study wassupported by CONACyT (grant: 38030-M); IMSS-FOFOI(FP-2001-053).

References

Aguilar, A., Camacho, J., Chino, S., Jaquez, P., Lopez, M.E., 1994.Herbario Medicinal del Instituto Mexicano del Seguro Social.I.M.S.S., Mexico, p. 43.

Alanis, A., Calzada, F., Cedillo-Rivera, R., Meckes, M., 2003. Antipro-tozoal activity of the constituents ofRubus coriifolius. PhytotherapyResearch 17, 681–682.

Amstrong, D., Cohen, J., 1999. Infectious Diseases, vol. 1, Section 2.Mosby, Spain, pp. 35.1–35.70 (Chapter 35).

Brown, J.W., 1979. Toxic megacolon associated with loperamide therapy.Journal of American Medicine Association 241, 501–502.

Caballero, G., Vanderheyden, P., Apers, S., Solis, P., Gupta, M., Pieters,L., Vauquelin, G., Vlietinck, A., 2003. In vitro effect of sanguinarinealkaloid on binding of [3H] candesartan to the human angiotensinaAT1 receptor. European Journal of Pharmacology 458, 257–262.

Caballero, G., Vanderheyden, P., Apers, S., Van den, H., Solis, P.,Gupta, M., Claeys, M., Pieters, L., Vauquelin, G., Vlietinck, A.,2002. Inhibitory activity on binding of specific ligands to the humanangiotensin II AT1 and endothelin 1 ETA receptors: bioactive benzo[c]

C from

8,

C tasal

D treat-32–

F new

Fischer, H., Machen, T., Widdicombe, J., Carlson, T., King, S., Chow, J.,Illek, B., 2004. A novel extract SB-300 from the stem bark latex ofCroton lechleri inhibits CFTR-mediated chloride secretion in humancolonic epithelial cells. Journal of Ethnopharmacology 93, 351–357.

Gabriel, S., Davenport, S., Steagall, R., Vimal, V., Carlson, T., Rozhon,E., 1999. A novel plant-derived inhibitor of cAMP-mediated fluid andchloride secretion. American Journal of Physiology 276, G58–G63.

Guerrant, R., 1985. Microbial toxins and diarrhoeal diseases. Introductionand overview. In: Evered, D., Whelan, J. (Eds.), Microbial Toxins andDiarrhoeal Disease, Ciba Fundation Symposium. London, pp. 1–5.

Gurgel, L., Silva, R., Santos, F., Martins, D., Mattos, P., Rao, V., 2001.Studies on the antidiarrhoeal effect of Dragon‘s Blood fromCrotonurucurana. Phytotherapy Research 15, 319–322.

Hor, M., Rimpler, H., Heinrich, M., 1995. Inhibition of intestinal chlo-ride secretion by proanthocyanidins fromGuazuma ulmifolia. PlantaMedica 61, 208–212.

Lara, F., Marquez, C., 1996. Plantas Medicinales de Mexico: Composicionusos y actividad biologica. UNAM, Mexico, p. 57.

Mutschler, E., Derendorf, H., Schafer, M., Elrod, K., Estes, K., 1995.Drugs Actions. CRC Press, Germany, pp. 434–436.

Perez, M., 2000. Actividad antibacteriana deBoconia frutescens. RevistaMexicana de Ciencias Farmaceuticas 31, 16–18.

Rabbani, G., Greenough III, Golmgren, J., Kirkwood, B., 1982. Controlledtrial of chlorpromazine as antisecretory agent in patients with cholerahydrated intravenously. British Medical Journal 284, 1361–1364.

Raufman, P., 1998. Cholera. American Journal of Medicine 104, 386–394.Roge, J., Baumer, P., Berard, H., Schwartz, J.C., Lecomte, J.M., 1993.

The enkephalinase inhibitor, acetorphan, in acute diarrhoea: a double-blind, controlled clinical trial versus loperamide. Scandinavian Journalof Gastroenterology 28, 352–354.

Sack, R., Froehlich, J., 1982. Berberine inhibits intestinal secretory-

S adotrilal of

T A.,

Tom7–11.

T G.,luble1.

phenanthridine alkaloids from the root ofBocconia frutescens. PlantaMedica 68, 770–775.

alzada, F., Cervantes, A., Yepez, L., 2005. Antiprotozoal activitythe roots ofGeranium mexicanum and its constituents onEntamoebahistolytica and Giardia lamblia. Journal of Ethnopharmacology 9191–193.

ampos, R., 1991. Estudios urbanos en Mexico sobre el uso de las planen Mexico. En: La investigacion cientıfica en la herbolaria medicinmexicana. Secretaria de Salud, Mexico, p. 138.

utta, N., Panse, V., 1962. Usefulness of berberine in thement of cholera. Indian Journal of Medicinal research 50, 7736.

edorak, R., Field, M., 1987. Antidiarrheal therapy prospects foragents. Digestive Diseases and Sciences 32, 195–205.

response ofVibrio cholerae and Escherichia coli enterotoxins. Infection and Immunity 35, 417–475.

alazar, E., Santisteban, J., Chea, E., Gutierrez, M., 2000. Racecin the treatment of acute watery diarrea. New England JournMedicine 343, 463–467.

orregosa, L., Santos, I., Rodrıguez, R., Velasquez, L., Garcia,Alpuche, C., 1996. Enfermedades diarreicas en el nino, 10th editionNew Interamerican Editorial, Mexico, pp. 41, 45–46.

orres, J., Lonnroth, I., Lange, S., Camorlinga, M., Gonzalez, S., Munoz,O., 1993. Antisecretory activity in a lecitin fraction of plasma frpatients with acute diarrhea. Archives of Medical Research 24,

urvill, L., Wapnir, A., Wingertzahn, A., Teichberg, S., Farthing,2000. Cholera toxin-induced secretion in rats is reduced by a sofiber, gum arabic. Digestive Diseases and Sciences 45, 946–95