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A study of the antidiarrheal properties of Loeselia mexicana on mice and rats.

Abstract

Antidiarrheal properties of hexane, chloroform, methanol and aqueous extracts from Loeselia mexicana were studied using mice and rats as animal models. Flavones and sesquiterpenlactones were detected in the chemical screening of the aqueous extract. Diarrhea was induced by castor oil or MgS[O.sub.4]. The methanol extract diminished diarrhea in mice induced by MgS[O.sub.4], while the aqueous extract showed an effect on castor-oil-induced. The aqueous extract also reduced castor-oil-enhanced intestinal transit and inhibited defecation of normal mice. The results obtained showed a symptomatic relief of diarrhea.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Loeselia mexicana; Antidiarrheal effect; Biologically active extracts

Introduction

Loeselia mexicana Lamb (Polemoniaceae), known commonly as "espinosilla" in Mexico, is an annual herb that grows to a height of 80 cm. It is found in warm climates at altitudes between 80 and 3900 m, and is used to treat gastrointestinal problems, to control fever and as an emetic (Argueta, 1992).

Previous investigations have revealed that an aqueous extract shows antidiarrheic activity in mice with castor-oil-induced diarrhea (Vargas et al., 1998), and an inhibitory effect on rat heart rates (Perez et al., 1998). Also, it was found that chloroform-methanol (1:1) extract produced an inhibition of spontaneous rat ileum contractions (Rojas et al., 1999). An alkaloid (loseline), tannins, saponins, essential oil (Martinez, 1967) and a pentacydic triterpenoid (Jimenez, 1989) have been reported as constituents.

In the present study, we evaluated the ability of hexane, chloroform, methanol and aqueous extracts, as well as the essential oil, from this plant to control diarrhea.

Methodology

Plant material

L. mexicana was obtained from the Sonora market in Mexico City in February 1998 and authenticated by M.S. Aurora Chimal of the Department "El Hombre y su Ambiente" of the Autonomous Metropolitana-Xochimilco University. A voucher specimen (GS31) was deposited in this herbarium. Aerial parts of the plant (leaves and branches) were shade-dried and powdered.

Preparation of the extracts

In a 1-1 bottom flask fitted with a reflux condenser, 100 g of dried powdered plant and 400 ml of solvent (hexane, chloroform, methanol or water) were refluxed for 4 h, cooled to room temperature and filtered. The hexane, chloroform and methanol extracts were dried in vacuo in a rotary evaporator and in a vacuum oven at 50[degrees]C for 12 h. The aqueous extract was lyophilized.

Extraction of essential oil

Fresh leaves were used and the essential oil was obtained by steam distillation, followed by extraction with ethyl ether. The yield was 0.22% v/w. The extracts and the essential oil were suspended in 10% Tween 80 and administered orally. Diphenoxylate (2.5 mg/kg body wt.) was used as reference standard.

Phytochemical screening

A preliminary screening of aqueous and methanol extracts showed positive Shinoda and Marini-Bettolo tests for flavones (Dominguez, 1979). It also showed positive Baljet and Legal reactions for sesquiterpenlactones (Dominguez, 1979), which were confirmed by thin-layer chromatography over silica gel G developed with chloroform-acetone 4:1 and with concentrated sulphuric acid and heated for 5 min at 100 [degrees]C.

Animals

CD1 strain male mice (20-25 g each) and male Wistar rats (200-250 g each) from the Autonomous Metropolitana University animal facility, fasted for 18-24 h, were housed in isolated cages under standard conditions (dark/light, 12/12) at 30[degrees]C and 50-55% humidity; they were supplied with purina chow and water ad libitum. The animals were fasted for 18-24 h with free access to water prior to the study.

Evaluation of the effect on normal defecation

Groups of five mice were placed individually in acrylic cages with filter paper at the bottom (Galvez et al., 1993). Each extract was administered to one group, diphenoxylate to another, and a third group served as vehicle-treated control. The total amount of feces in each group was assessed every hour for the next 3h. The percentage reduction in the amount of feces in the treated group was obtained by comparison with control animals (0% reduction).

Evaluation of antidiarrheal activity

Either an extract or diphenoxylate was administered to groups of five mice 30 min before the administration of cathartic agent (castor oil 4 ml/kg or Mg[SO.sub.4] 2.0 g/kg body wt.) (Melo et al., 1988). Following treatment, the animals were placed separately in acrylic cages with filter paper at the bottom, which was changed every hour. The severity of diarrhea was assessed each hour for 4 h, on an arbitrary, depending on the consistency and the number of feces present on each paper, as follows; 0 = no feces; 1 = normal feces; 2 = soft feces; 3 = watery feces. The total number of watery feces excreted was scored and compared with the score from the control group. The total score of diarrheic feces of the control group was considered to be 100%. The results were expressed as a percentage of inhibition.

Small intestinal transit

For the evaluation of small intestinal transit, the method described by Visher and Casals-Stenzel (1982) was followed. A 2.0% suspension of graphite in 1.5% agar was administered orally to groups of 15 rats (1.5 ml/animal). The inhibitory action of the extracts on stimulated intestinal transit was tested using the following procedure. Castor oil (4 ml/kg body wt.) was administered, along with the graphite-agar suspension 60 min after the vehicle or the extract. At 30, 60 and 90 min after the administration of a graphite-agar suspension plus castor oil, the rats were killed in groups of five and the gastrointestinal tract was removed and opened. The distance traveled by the marker was measured and expressed as a percentage of the total length of the intestine from pylorus to caecum. The mean value for each group was calculated and the results obtained in the control and test groups were compared. To obtain the percentage of inhibition, the following equation was applied:

% of inhibition = [(mean distance in control group - mean distance in treated group)/[mean distance in control group]] X 100.

Antimicrobial screening

An antimicrobial test was performed using brain-heart infusion with bacteriology agar (1.5%) (Ronald, 1990). Nutrient media was prepared according to manufacturer's instructions and sterilized for 15 min at 120[degrees]C. For the determination of antimicrobial activity, the standard method of disc diffusion plates on agar was used. The following microorganisms were used: Escherichia coli ATCC 10586; E. coli (EHEC); Salmonella choleraesuis ATCC 10708; Salmonella typhimurium ATCC 14028; and Vibrio cholerae 01 ATCC 51328. Bacteria were incubated for 24 h at 37[+ or -]1[degrees]C. Each culture was prepared to a turbidity equivalent to McFarland tube Number 4; I ml of culture was spread on the test plate. Four blank paper disks (6 mm in diameter) containing 1.0 mg of the plant extract were placed on the test plate. All determinations were made in duplicate. Ampicillin (2 [micro]g) or chloramphenicol (30 [micro]g) was used as positive control. Sensidiscs were obtained from BBL. Inhibition diameters were read after incubation, and measured in mm.

Statistics

Results are expressed as mean [+ or -] s.e.m. Mean values of intestinal transit were evaluated by Student's t-test, while a non-parametric Kruskal-Wallis test was used to compare the mean values of the antidiarrheal activity with the control group. Statistical significance was accepted at p < 0.05.

Results

L. mexicana is used commonly for the treatment of symptoms of diarrhea. Since antidiarrheal effects may be due to many compounds, we evaluated the pharmacological effect of the essential oil and the hexane, chloroform, methanol and aqueous extracts of this plant.

The chemical screening of this plant showed the presence of flavones and sesquiterpene lactones, in addition to those reported, including loseline, tannins and saponins.

The results of evaluation of the extracts showed that hexane and chloroform extracts had no effect on animals in the pharmacological models used in this work; however, the aqueous extract diminished castor-oil-induced diarrhea, while the methanol extract inhibited MgS[O.sub.4]-induced diarrhea. The frequency of wet feces was not diminished with the essential oil (50 mg/kg body wt.).

The activity of the aqueous extract was not dose-dependent. The average effect ranged from 23% to 34% of inhibition at doses of 12.5, 25 and 50 mg/kg body wt. At doses of 100 mg/kg body wt., however, inhibition was not observed (Table 1). This extract did not have any effect on mice with MgS[O.sub.4]-induced diarrhea. The aqueous extract inhibited the defecation of normal mice (Table 2). The major effect was observed at doses of 100 mg/kg body wt. (92.3%).

The pattern of intestinal transit in normal rats and in castor-oil-treated rats, together with the effect of aqueous extract at doses of 50 mg/kg body wt. on castor oil-stimulated intestinal transit, is shown in Fig. 1. During the first 60 min, aqueous extract reduced intestinal transit even below that of normal values. At 90 min, there were no significant differences between the control and aqueous-extract-treated rats which received castor oil.

When diarrhea was induced using MgS[O.sub.4], the major effect of the methanol extract was observed at doses of 100 mg/kg body wt. (44.4%), although at doses of 200 mg/kg body wt. a slight decrease of the effect was observed (28.9%) (Table 3). In addition, this extract did not have any effect on mice with castor-oil-induced diarrhea. This extract did not modify the pattern of intestinal transit (Fig. 1) and did not have any activity on the defecation of normal mice (Table 2).

[FIGURE 1 OMITTED]

Discussion

Castor oil increases peristaltic activity of the intestine. Moreover, it has been associated with mucosal injury (Awouters et al., 1975) and the liberation of several endogenous mediators such as prostaglandins (Pons et al., 1994; Pinto et al., 1992) and nitric oxide (Mascolo et al., 1994).

The osmotic properties of MgS[O.sub.4] prevent the reabsorption of water and ions, leading to an increase in the volume of the intestinal content. This compound also promotes the production of cholecystokinin from duodenal mucosa, which further increases the secretions and has a positive motor effect on the small intestine (Galvez et al., 1993). The methanol extract inhibited the effect of this cathartic agent. Given the results, it seems reasonable to suppose that the antidiarrheal effect of the aqueous extract might be due to one of the factors mentioned previously. In addition, this extract appears to act on all parts of the intestine. It inhibited castor-oil-stimulated small intestinal transit 60 min after administration.

The antidiarrheal activity of the aqueous and methanol extracts might be due to different compounds because their effects in the animal models are different.

It has been found in some cases that the antidiarrheal effect is associated with other pharmacological effects; in this case, we evaluated the antimicrobial activity (Caceres et al., 1993) on Gram (-) and Gram (+) microorganisms. None of the extracts from L. mexicana showed antimicrobial activity against the bacteria used, and the antidiarrheal effect cannot therefore be associated to this activity.

It can be concluded that the present study supports the claims by traditional medicine practitioners about the value of L. mexicana for the treatment of diarrhea.

Alkaloids, tannins, saponins, flavones, glycosides and sesquiterpenlactones have been identified from this plant. Because many of these compounds might have antidiarrheal effects, it is difficult to suggest which of them is responsible for the effect observed. The phytochemical study of this plant will continue in order to identify the compounds responsible for this activity.

Acknowledgement

The authors are thankful to M.S. Aurora Chimal Hernandez for the authentication of plant material and Michael Shea for technical assistance. This work was funded partly by financial aid from CONACYT (project B-10304).

References

Argueta, V.A., 1992. Atlas de las Plantas Medicinales de Mexico Instituto Nacional Indigenista. Mexico DF.

Awouters, F., Niemegeers, C.J.E., Kuyps, J., Janssen, P.A.J., 1975. Loperamide antagonism of castor oil-induced diarrhea in rats: a quantitative study. Arch. Int. Pharmacodyn. 217, 29-37.

Caceres, A., Fletes, L., Aguilar, L., Ramirez, O., Figueroa, L., Taracena, A.M., Samayoa, B., 1993. Plants used in Guatemala for the treatment of gastrointestinal disorders, 3. Confirmation of activity against enterobacteria of 16 plants. J. Ethnopharmacol. 38, 31-38.

Domiinguez, X.A., 1979. Metodos de Investigacion. Fitoquimica Editorial Limusa, Mexico DF, pp. 84-97.

Galvez, J., Zarzuelo, A., Crespo, M.E., 1993. Antidiarrhoeic activity of Euphorbia hirta extract and isolation of an active flavonoid. Planta Med. 59, 333-336.

Jimenez, M., 1989. Structure of a pentacyclic triterpenyl angelate from Loeselia mexicana H2D NMR data and stereochemistry. Can. J. Chem. 67 (2), 2071-2077.

Martinez, M., 1967. Las Plantas Medicinales de Mexico, sixth ed. Editorial Botas, Mexico DF, pp. 131-133.

Mascolo, N., Izzo, A.A., Autore, G., Barbato, F., Capasso, F., 1994. Nitric oxide and castor oil-induced diarrhea. J. Pharmacol. Exp. Ther. 268 (1), 291-295.

Melo, L., Thomas, G., Mukherjee, R., 1988. Antidiarrheal activity of bisnordihydroxytoxiferine isolated from the root bark of Strychnos trinervis (Vell) Mart (Loganiaceae). J. Pharm. Pharmacol. 40, 79-82.

Perez, G.S., Vargas, S.R., Zavala, S.M., Perez, G.C., 1998. Inhibitory effect of five plant extracts on heart rates of rats. Phytother. Res. 12, S49-S50.

Pinto, A., Calignano, A., Mascolo, N., Sorrentino, R., Biondi, A., Izzo, A.A., Capasso, F., 1992. Time course of PAF formation by gastrointestinal tissue in rats after castor oil challenge. J. Pharm. Pharmacol. 44, 224-226.

Pons, L., Droy-Lefaix, M.T., Bueno, L., 1994. Role of platelet-activating factor (PAF) and prostaglandins in colonic motor and secretory disturbances induced by Escherichia coli endotoxin in conscious rats. Prostaglandins 91, 947-951.

Rojas, A., Bah, M., Rojas, J.I., Serrano, V., Pacheco, S., 1999. Spasmolytic activity of some plants used by the Otomi Indians of Queretaro (Mexico) for the treatment of gastrointestinal disorders. Phytomedicine 6, 367-371.

Ronald, M.A., 1990. Microbiologia Compania. Editorial Continental SA de CV, Mexico DF, p. 505.

Vargas, S.R., Zavala, S.M., Perez, G.C., Perez, G.R.M., Perez, G.S., 1998. Preliminary study of antidiarrheic activity in five Mexican plant species. Phytother. Res. 12, S47-S48.

Visher, P., Casals-Stenzel, J., 1982. Influence of prostacyclin and indomethacin on castor oil induced gastrointestinal effects in rats. J. Pharm. Pharmacol. 35, 152-155.

Salud Perez G., Cuauhtemoc Perez G., M.A. Zavala S.*

Departamento de Sistemas Biologicos, Universidad Autonoma Metropolitana-Xochimilco, Calzada del Hueso 1100, Col. Villa Quietud, Coyoacan, Mexico D.F., Mexico

Received 12 December 2003; accepted 25 January 2004

*Corresponding author. Tel./fax: 52 55 54837410.

E-mail address: mzavala@cueyatl.uam.mx (M.A. Zavala S.).
Table 1. Antidiarrheal activity of the aqueous extract of L. mexicana on
mice with castor-oil-induced diarrhea (a)

 Doses (mg/ Percent
Treatment kg body wt., p.o.) inhibition (b)

Vehicle 0
Extract 12.5 23.1 [+ or -] 1.44*
 25.0 22.2 [+ or -] 1.9*
 50 34.1 [+ or -] 0.75**
 100 0
 200 0
Diphenoxylate 2.5 97.8 [+ or -] 0.89

(a) Results are the mean of 20 animals [+ or -] s.e.m.
(b) Significant difference was found: *p < 0.05; **p < 0.01.

Table 2. Effect of different extracts on normal defecation of mice (a)

 Doses (mg/k[g.sup.-1] Percent inhibition (b)
Plant Treatment body wt., p. o.)

L. mexicana Aqueous 50 4.61 [+ or -] 4.47**
 extract
 100 92.3 [+ or -] 5.44**
L. mexicana Methanol 50 3.1 [+ or -] 1.22 (NS)
 extract
 100 4.8 [+ or -] 2.14 (NS)

(a) Results are the mean of 20 animals [+ or -] s.e.m.
(b) Significant difference was found: **p < 0.01; NS, no significant
difference.

Table 3. Antidiarrheal activity of the methanol extract of Loeselia
mexicana on mice with MgS[O.sub.4]-induced diarrhea (a)

 Doses (mg/ Percent
Treatment kg body wt., p.o) inhibition (b)

Vehicle 0
Extract 12.5 18.1 [+ or -] 5.7 (NS)
 25.0 29.17 [+ or -] 6.4*
 50 33.14 [+ or -] 4.0*
 100 44.44 [+ or -] 3.8**
 200 28.93 [+ or -] 2.2*
Diphenoxylate 2.5 100

(a) Results are the mean of 20 animals [+ or -] s.e.m.
(b) Significant difference was found: *p < 0.05; **p < 0.01; NS, no
significant difference.
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Author:Perez G., Salud; Perez G., Cuauhtemoc; Zavala S., M.A.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Sep 1, 2005
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