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In vivo antigiardial activity of three flavonoids isolated of some medicinal plants used in Mexican traditional medicine for the treatment of diarrhea
Journal of Ethnopharmacology 109 (2007) 552–554
Ethnopharmacological communication
In vivo antigiardial activity of three flavonoids isolated of some medicinal plants used in Mexican traditional medicine for the treatment of diarrhea夽 Elizabeth Barbosa a,b,∗ , Fernando Calzada a,∗ , Rafael Campos b a
Unidad de Investigaci´on Medica en Farmacolog´ıa de Productos Naturales, Hospital de Pediatr´ıa, 2 Piso, Centro Medico Nacional Siglo XXI, IMSS, Av. Cuauhtemoc 330, Col. Doctores, CP 06725, M´exico, D.F., M´exico b Departamento de Bioqu´ımica, Instituto Polit´ ecnico Nacional, Escuela Superior de Medicina, Plan de San Luis y D´ıaz Mir´on, CP 11340, M´exico, D.F., M´exico Received 7 June 2006; received in revised form 8 September 2006; accepted 11 September 2006 Available online 16 September 2006
Abstract Mexican traditional medicine uses a great variety of plants in the treatment of gastrointestinal disorders such as diarrhea. In order to understand the properties of some of their chemical constituents, three flavonoids (kaempferol, tiliroside and (−)-epicatechin) isolated from Geranium mexicanum, Cuphea pinetorum, Helianthemum glomeratum, and Rubus coriifolius, were assayed to demonstrate their in vivo antiprotozoal activity; using an experimental infection of Giardia lamblia in suckling female CD-1 mice. Compounds tested showed antigiardial activity with values of ED50 (mol/kg) 0.072 for (−)-epicatechin, 2.057 for kaempferol and 1.429 for tiliroside. The most active flavonoid was the (−)-epicatechin, its activity was higher than metronidazole and emetine, drugs used as positive controls. In the case of kaempferol and tiliroside their potency was close to that of the metronidazole, but far less than emetine. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Medicinal plants; Flavonoids; Kaempferol; Tiliroside; (−)-Epicatechin; Giardia lamblia; Antigiardial activity
1. Introduction Diarrheal infection diseases are a great problem throughout the world and responsible for considerable morbidity and mortality, especially in developing countries (Hlavsa et al., 2005). In Mexico giardiasis is the most prevalent parasitic disease in 1–9-year-old children (Jim´enez et al., 2003). In Mexico traditional medicine uses a great variety of plants in the treatment of gastrointestinal disorders such as diarrhea, caused in some cases by Giardia lamblia. In this sense, from the species Geranium mexicanum HBK (Geraniaceae), Cuphea pinetorum Benth (Litraceae), Helianthemum glomeratum Lag. (Cistaceae), and Rubus coriifolius Focke (Rosaceae), compounds with in vitro antigiardial properties have been isolated, these compounds were identified as flavonoids kaempferol, tiliroside and (−)-epicatechin (Calzada et al., 1998, 1999, 2005; Alan´ıs et al., 2003; Calzada, 2005).
Flavonoids have been reported to exert multiple biological effects due to their antioxidant and free radical-scavenging abilities; although results from different studies have demonstrated that flavonoids can act as pro-oxidant at very high doses. Furthermore most investigations have reported antibacterial, antifungal, antiviral, anti-inflammatory, anti-allergic activity and protective roles in heart diseases, cancer and different pathologies (Mart´ınez et al., 2002; Matsuda et al., 2003). As related in these data, in this paper we report the in vivo antigiardial activity of flavonoids, kaempferol, tiliroside and (−)-epicatechin, using an experimental infection of Giardia lamblia in suckling female CD-1 mice, therefore the results will provide important information to give continuity to the study of flavonoids as prototype molecules for the development of new drugs useful in the treatment of giardiasis. 2. Materials and methods 2.1. Isolation of test compounds
夽 ∗
Part of this work was taken from the Ph.D. thesis of Elizabeth Barbosa. Corresponding authors. Tel.: +52 5 627 6900x22410; fax: +52 5 761 0952. E-mail address: [email protected] (F. Calzada).
0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.09.009
The test flavonoids were isolated from different plant species: thus kaempferol and tiliroside were obtained from Cuphea
E. Barbosa et al. / Journal of Ethnopharmacology 109 (2007) 552–554
pinetorum Benth (Voucher 9273) and Helianthemum glomeratum Lag. (Voucher 14142). In the case of (−)-epicatechin, it was obtained from Rubus coriifolius Focke (Voucher 11874) and Geranium mexicanum HBK (Voucher Calzada 14405), the extraction and isolation procedures of the flavonoids were done as it has been described previously (Calzada et al., 1998, 1999, 2005; Alan´ıs et al., 2003; Calzada, 2005). The plants were collected from different regions of Mexico. Voucher specimens were deposited at the Herbarium IMSSM of the Instituto Mexicano del Seguro Social (IMSS).
553
Table 1 In vivo activity of flavonoids against Giardia lamblia WB in CD-1 micea Compound
ED50 (mol/kg) (CI)b
Kaempferol Tiliroside (−)-Epicatechin Metronidazolec Emetinec
2.057 (2.075–2.043) 1.429 (1.452–1.405) 0.072 (0.076–0.072) 1.134 (1.140–1.122) 0.351 (0.353–0.349)
a b c
Results are expressed as mean (n = 6). Dose required to kill 50% of the organisms with (IC) 95% confidence limits. Positive control.
2.2. Cultures Giardia lamblia strain WB was grown axenically at 37 ◦ C in TYI-S-33 medium supplemented with bile salts and 10% heat inactivated bovine serum (Keister, 1983; Cedillo et al., 1991).
the trophozoites (ED50 ) together with the 95% confidence limits were computed from a plot of probit against the logarithm of the drug concentration (Finney, 1977). 3. Results and discussion
2.3. Antigiardial activity The in vivo antigiardial activity of the extracts was tested using a method previously described (Roberts et al., 1976; Belosevic and Faubert, 1983; Boreham et al., 1986; Reynoldson et al., 1991). Female CD-1 mice (16–24 g) were obtained of the animal house in Escuela Superior de Medicina from IPN. Animals were kept under standard conditions. The experimental protocols were approved by the Animal Care and Use Committee of Hospital de Pediatria del Centro Medico Nacional Siglo XXI, IMSS, in accordance with the guidelines for care and use of laboratory animals. The effect of the flavonoids was studied on mice infected with trophozoites of Giardia lamblia. Mice were treated for three consecutive days with mebendazole solution (10 mg/mouse/day), which was administered orally. This treatment ensured that mice were free from all possible protozoan infections. One week after treatment, three consecutive fecal examinations were performed prior to experimental infections. Mice (n = 6 per group) were infected intragastrically with 1 × 106 trophozoites in 300 l of TYI-S-33 medium via a silastic tube (0.0025 in. outside diameter) attached to a 1 ml syringe. Six days after infection, mice were treated intragastrically with 300 l of the flavonoid under study, dissolved in 1 ml of a 2% dimethyl sulphoxide (DMSO) solution in water. The control group received the vehicle alone (2% DMSO solution in water). Forty-eight hours after flavonoid treatment (8 days postinfection) the mice were killed and the entire small intestine removed, opened longitudinally and placed in 10 ml of a 10% PAF fixer for a least 2 h. Tubes were vortexed to ensure complete detachment of the trophozoites and the number of organisms present calculated for both the treated and control groups. Five single-doses of each flavonoid were tested. Metronidazole and emetine drugs were used as positive controls.
Traditional Mexican medicine uses a wide variety of plants in the treatment of diarrhea. In this sense of the species Geranium mexicanum, Cuphea pinetorum, Helianthemum glomeratum, and Rubus coriifolius have demonstrated in vitro antigiardial properties, as well as the isolated flavonoids from these plants (Calzada et al., 1998, 1999, 2005; Alan´ıs et al., 2003; Calzada, 2005). The antiprotozoal activity of these flavonoids was tested using an experimental infection with Giardia lamblia in animal model. Table 1 shows the ED50 values for the compound assessed. The three flavonoids show in vivo activity against Giardia lamblia, (−)-epicatechin was the most active being 30 and 40 times more active than kaempferol and tiliroside, in relation with drugs used as positive controls it was 10 and 8 times more active than metronidazole and emetine, respectively. Kaempferol and tiliroside were equipotent with metronidazole and were less active than emetine. Data obtained in this investigation, combined with our previous results confirm that kaempferol, tiliroside, and (−)epicatechin, may be used as leading compounds for the development of novel therapeutic drugs for the treatment of diarrhea. Furthermore, these results are consistent with those described previously in relation to the in vitro activity, where (−)epicatechin was the most active flavonoid (Calzada et al., 1999; Alan´ıs et al., 2003). Also, and due to medicinal potential of crude extracts or fractions of Cuphea pinetorum, Helianthemum glomeratum, Rubus coriifolius, and Geranium mexicanum, they can be used for the phytodrugs development. The presence of the antigiardial compounds, kaempferol, tiliroside, and (−)-epicatechin in several plant species used traditionally for the treatment of diarrhea could account for the effectiveness of such plants in folk medicine as therapeutic agents. 4. Conclusions
2.4. Statistical analysis Data were analyzed using probit analysis. The percentage of trophozoites surviving was calculated by comparison with the growth in the control group. The dose required to kill 50% of
The results of this study provide important information to give continuity to the search of new antiprotozoal agents and to propose the flavonoids as prototype molecules for the development of new drugs against Giardia lamblia. This is the first
554
E. Barbosa et al. / Journal of Ethnopharmacology 109 (2007) 552–554
report on flavonoids kaempferol, tiliroside and (−)-epicatechin about their in vivo antiprotozoal activities. Consequently the toxicological studies to define the potential therapeutic benefit of these flavonoids will be carried out. References Alan´ıs, A., Calzada, F., Cedillo, R., Meckes, M., 2003. Antiprotozoal activity of the constituents of Rubus coriifolius. Phytotherapy Research 17, 681–682. Belosevic, M., Faubert, G., 1983. Giardia muris: correlation between oral dosage, course of infection, and trophozoite distribution in the mouse small intestine. Experimental Parasitology 56, 93–100. Boreham, P., Phillips, R., Shepherd, R., 1986. The activity of drugs against Giardia intestinalis in neonatal mice. Journal of Antimicrobial Chemotherapy 18, 393–398. Calzada, F., 2005. Additional antiprotozoal constituents from Cuphea pinetorum, a plant used in Mayan traditional medicine to treat diarrhoea. Phytotherapy Research 19, 725–727. Calzada, F., Alan´ıs, A., Meckes, M., Tapia, A., Cedillo, R., 1998. In vitro susceptibility of Entamoeba histolytica and Giardia lamblia to some medicinal plants used by the people of southern Mexico. Phytotherapy Research 12, 70–72. Calzada, F., Cervantes, J., Yepez, L., 2005. In vitro antiprotozoal activity from the roots of Geranium mexicanum and its constituents on Entamoeba histolytica and Giardia lamblia. Journal of Ethnopharmacology 98, 191–193.
Calzada, F., Meckes, M., Cedillo, R., 1999. Antiamoebic and antigiardial activity of plant flavonoids. Planta Medica 65, 78–80. Cedillo, R., Enciso, J., Mart´ınez, A., Ortega, G., 1991. Isolation and axenization of Giardia lamblia isolates from symptomatic and asymptomatic patients in Mexico. Archives of Medical Research 22, 79–85. Finney, D.L., 1977. Probit Analysis. Cambridge University Press, New York, p. 20. Hlavsa, M., Watson, J., Beach, M., 2005. Giardiasis Surveillance – United States, 1998–2002. Morbidity and Mortality Weekly Report. Surveillance Summaries 54 (SS1), 9–16. Jim´enez, E., Flores, A., Angeles, E., Mart´ınez, P., L´opez, R., Casta˜neda, G., P´erez, J., 2003. In vitro antigiardial activity of IRE-6A and IRE-7B, two ethyl-phenylcarbamate derivatives. Revista de Investigaci´on Cl´ınica 55, 444–447. Keister, D.B., 1983. Axenic culture of Giadia lamblia in TYI-S-33 medium supplemented with bile. Transactions of the Royal Society of Tropical Medicine and Hygiene 77, 487–488. Mart´ınez, S., Gonz´alez, J., Culebras, J., Mu˜no´ n, M., 2002. Los flavonoides: propiedades y acciones antioxidantes. Nutrici´on Hospitalaria 17, 271–278. Matsuda, H., Morikawa, T., Ando, S., Toguchida, I., Yoshikawa, M., 2003. Structural requirements of flavonoids for nitric oxide production inhibitory activity and mechanism of action. Bioorganic and Medicinal Chemistry 11, 1995–2000. Reynoldson, J., Thompson, R., Meloni, B., 1991. In vivo efficacy of albendazole against Giaria duodenalis in mice. Parasitology Research 77, 325–328. Roberts, I., Stevens, D., Mahmoud, A., Warren, K., 1976. Giardiasis in the mouse: an animal model. Gastroenterology 71, 57–61.
Ethnopharmacological communication
In vivo antigiardial activity of three flavonoids isolated of some medicinal plants used in Mexican traditional medicine for the treatment of diarrhea夽 Elizabeth Barbosa a,b,∗ , Fernando Calzada a,∗ , Rafael Campos b a
Unidad de Investigaci´on Medica en Farmacolog´ıa de Productos Naturales, Hospital de Pediatr´ıa, 2 Piso, Centro Medico Nacional Siglo XXI, IMSS, Av. Cuauhtemoc 330, Col. Doctores, CP 06725, M´exico, D.F., M´exico b Departamento de Bioqu´ımica, Instituto Polit´ ecnico Nacional, Escuela Superior de Medicina, Plan de San Luis y D´ıaz Mir´on, CP 11340, M´exico, D.F., M´exico Received 7 June 2006; received in revised form 8 September 2006; accepted 11 September 2006 Available online 16 September 2006
Abstract Mexican traditional medicine uses a great variety of plants in the treatment of gastrointestinal disorders such as diarrhea. In order to understand the properties of some of their chemical constituents, three flavonoids (kaempferol, tiliroside and (−)-epicatechin) isolated from Geranium mexicanum, Cuphea pinetorum, Helianthemum glomeratum, and Rubus coriifolius, were assayed to demonstrate their in vivo antiprotozoal activity; using an experimental infection of Giardia lamblia in suckling female CD-1 mice. Compounds tested showed antigiardial activity with values of ED50 (mol/kg) 0.072 for (−)-epicatechin, 2.057 for kaempferol and 1.429 for tiliroside. The most active flavonoid was the (−)-epicatechin, its activity was higher than metronidazole and emetine, drugs used as positive controls. In the case of kaempferol and tiliroside their potency was close to that of the metronidazole, but far less than emetine. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Medicinal plants; Flavonoids; Kaempferol; Tiliroside; (−)-Epicatechin; Giardia lamblia; Antigiardial activity
1. Introduction Diarrheal infection diseases are a great problem throughout the world and responsible for considerable morbidity and mortality, especially in developing countries (Hlavsa et al., 2005). In Mexico giardiasis is the most prevalent parasitic disease in 1–9-year-old children (Jim´enez et al., 2003). In Mexico traditional medicine uses a great variety of plants in the treatment of gastrointestinal disorders such as diarrhea, caused in some cases by Giardia lamblia. In this sense, from the species Geranium mexicanum HBK (Geraniaceae), Cuphea pinetorum Benth (Litraceae), Helianthemum glomeratum Lag. (Cistaceae), and Rubus coriifolius Focke (Rosaceae), compounds with in vitro antigiardial properties have been isolated, these compounds were identified as flavonoids kaempferol, tiliroside and (−)-epicatechin (Calzada et al., 1998, 1999, 2005; Alan´ıs et al., 2003; Calzada, 2005).
Flavonoids have been reported to exert multiple biological effects due to their antioxidant and free radical-scavenging abilities; although results from different studies have demonstrated that flavonoids can act as pro-oxidant at very high doses. Furthermore most investigations have reported antibacterial, antifungal, antiviral, anti-inflammatory, anti-allergic activity and protective roles in heart diseases, cancer and different pathologies (Mart´ınez et al., 2002; Matsuda et al., 2003). As related in these data, in this paper we report the in vivo antigiardial activity of flavonoids, kaempferol, tiliroside and (−)-epicatechin, using an experimental infection of Giardia lamblia in suckling female CD-1 mice, therefore the results will provide important information to give continuity to the study of flavonoids as prototype molecules for the development of new drugs useful in the treatment of giardiasis. 2. Materials and methods 2.1. Isolation of test compounds
夽 ∗
Part of this work was taken from the Ph.D. thesis of Elizabeth Barbosa. Corresponding authors. Tel.: +52 5 627 6900x22410; fax: +52 5 761 0952. E-mail address: [email protected] (F. Calzada).
0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.09.009
The test flavonoids were isolated from different plant species: thus kaempferol and tiliroside were obtained from Cuphea
E. Barbosa et al. / Journal of Ethnopharmacology 109 (2007) 552–554
pinetorum Benth (Voucher 9273) and Helianthemum glomeratum Lag. (Voucher 14142). In the case of (−)-epicatechin, it was obtained from Rubus coriifolius Focke (Voucher 11874) and Geranium mexicanum HBK (Voucher Calzada 14405), the extraction and isolation procedures of the flavonoids were done as it has been described previously (Calzada et al., 1998, 1999, 2005; Alan´ıs et al., 2003; Calzada, 2005). The plants were collected from different regions of Mexico. Voucher specimens were deposited at the Herbarium IMSSM of the Instituto Mexicano del Seguro Social (IMSS).
553
Table 1 In vivo activity of flavonoids against Giardia lamblia WB in CD-1 micea Compound
ED50 (mol/kg) (CI)b
Kaempferol Tiliroside (−)-Epicatechin Metronidazolec Emetinec
2.057 (2.075–2.043) 1.429 (1.452–1.405) 0.072 (0.076–0.072) 1.134 (1.140–1.122) 0.351 (0.353–0.349)
a b c
Results are expressed as mean (n = 6). Dose required to kill 50% of the organisms with (IC) 95% confidence limits. Positive control.
2.2. Cultures Giardia lamblia strain WB was grown axenically at 37 ◦ C in TYI-S-33 medium supplemented with bile salts and 10% heat inactivated bovine serum (Keister, 1983; Cedillo et al., 1991).
the trophozoites (ED50 ) together with the 95% confidence limits were computed from a plot of probit against the logarithm of the drug concentration (Finney, 1977). 3. Results and discussion
2.3. Antigiardial activity The in vivo antigiardial activity of the extracts was tested using a method previously described (Roberts et al., 1976; Belosevic and Faubert, 1983; Boreham et al., 1986; Reynoldson et al., 1991). Female CD-1 mice (16–24 g) were obtained of the animal house in Escuela Superior de Medicina from IPN. Animals were kept under standard conditions. The experimental protocols were approved by the Animal Care and Use Committee of Hospital de Pediatria del Centro Medico Nacional Siglo XXI, IMSS, in accordance with the guidelines for care and use of laboratory animals. The effect of the flavonoids was studied on mice infected with trophozoites of Giardia lamblia. Mice were treated for three consecutive days with mebendazole solution (10 mg/mouse/day), which was administered orally. This treatment ensured that mice were free from all possible protozoan infections. One week after treatment, three consecutive fecal examinations were performed prior to experimental infections. Mice (n = 6 per group) were infected intragastrically with 1 × 106 trophozoites in 300 l of TYI-S-33 medium via a silastic tube (0.0025 in. outside diameter) attached to a 1 ml syringe. Six days after infection, mice were treated intragastrically with 300 l of the flavonoid under study, dissolved in 1 ml of a 2% dimethyl sulphoxide (DMSO) solution in water. The control group received the vehicle alone (2% DMSO solution in water). Forty-eight hours after flavonoid treatment (8 days postinfection) the mice were killed and the entire small intestine removed, opened longitudinally and placed in 10 ml of a 10% PAF fixer for a least 2 h. Tubes were vortexed to ensure complete detachment of the trophozoites and the number of organisms present calculated for both the treated and control groups. Five single-doses of each flavonoid were tested. Metronidazole and emetine drugs were used as positive controls.
Traditional Mexican medicine uses a wide variety of plants in the treatment of diarrhea. In this sense of the species Geranium mexicanum, Cuphea pinetorum, Helianthemum glomeratum, and Rubus coriifolius have demonstrated in vitro antigiardial properties, as well as the isolated flavonoids from these plants (Calzada et al., 1998, 1999, 2005; Alan´ıs et al., 2003; Calzada, 2005). The antiprotozoal activity of these flavonoids was tested using an experimental infection with Giardia lamblia in animal model. Table 1 shows the ED50 values for the compound assessed. The three flavonoids show in vivo activity against Giardia lamblia, (−)-epicatechin was the most active being 30 and 40 times more active than kaempferol and tiliroside, in relation with drugs used as positive controls it was 10 and 8 times more active than metronidazole and emetine, respectively. Kaempferol and tiliroside were equipotent with metronidazole and were less active than emetine. Data obtained in this investigation, combined with our previous results confirm that kaempferol, tiliroside, and (−)epicatechin, may be used as leading compounds for the development of novel therapeutic drugs for the treatment of diarrhea. Furthermore, these results are consistent with those described previously in relation to the in vitro activity, where (−)epicatechin was the most active flavonoid (Calzada et al., 1999; Alan´ıs et al., 2003). Also, and due to medicinal potential of crude extracts or fractions of Cuphea pinetorum, Helianthemum glomeratum, Rubus coriifolius, and Geranium mexicanum, they can be used for the phytodrugs development. The presence of the antigiardial compounds, kaempferol, tiliroside, and (−)-epicatechin in several plant species used traditionally for the treatment of diarrhea could account for the effectiveness of such plants in folk medicine as therapeutic agents. 4. Conclusions
2.4. Statistical analysis Data were analyzed using probit analysis. The percentage of trophozoites surviving was calculated by comparison with the growth in the control group. The dose required to kill 50% of
The results of this study provide important information to give continuity to the search of new antiprotozoal agents and to propose the flavonoids as prototype molecules for the development of new drugs against Giardia lamblia. This is the first
554
E. Barbosa et al. / Journal of Ethnopharmacology 109 (2007) 552–554
report on flavonoids kaempferol, tiliroside and (−)-epicatechin about their in vivo antiprotozoal activities. Consequently the toxicological studies to define the potential therapeutic benefit of these flavonoids will be carried out. References Alan´ıs, A., Calzada, F., Cedillo, R., Meckes, M., 2003. Antiprotozoal activity of the constituents of Rubus coriifolius. Phytotherapy Research 17, 681–682. Belosevic, M., Faubert, G., 1983. Giardia muris: correlation between oral dosage, course of infection, and trophozoite distribution in the mouse small intestine. Experimental Parasitology 56, 93–100. Boreham, P., Phillips, R., Shepherd, R., 1986. The activity of drugs against Giardia intestinalis in neonatal mice. Journal of Antimicrobial Chemotherapy 18, 393–398. Calzada, F., 2005. Additional antiprotozoal constituents from Cuphea pinetorum, a plant used in Mayan traditional medicine to treat diarrhoea. Phytotherapy Research 19, 725–727. Calzada, F., Alan´ıs, A., Meckes, M., Tapia, A., Cedillo, R., 1998. In vitro susceptibility of Entamoeba histolytica and Giardia lamblia to some medicinal plants used by the people of southern Mexico. Phytotherapy Research 12, 70–72. Calzada, F., Cervantes, J., Yepez, L., 2005. In vitro antiprotozoal activity from the roots of Geranium mexicanum and its constituents on Entamoeba histolytica and Giardia lamblia. Journal of Ethnopharmacology 98, 191–193.
Calzada, F., Meckes, M., Cedillo, R., 1999. Antiamoebic and antigiardial activity of plant flavonoids. Planta Medica 65, 78–80. Cedillo, R., Enciso, J., Mart´ınez, A., Ortega, G., 1991. Isolation and axenization of Giardia lamblia isolates from symptomatic and asymptomatic patients in Mexico. Archives of Medical Research 22, 79–85. Finney, D.L., 1977. Probit Analysis. Cambridge University Press, New York, p. 20. Hlavsa, M., Watson, J., Beach, M., 2005. Giardiasis Surveillance – United States, 1998–2002. Morbidity and Mortality Weekly Report. Surveillance Summaries 54 (SS1), 9–16. Jim´enez, E., Flores, A., Angeles, E., Mart´ınez, P., L´opez, R., Casta˜neda, G., P´erez, J., 2003. In vitro antigiardial activity of IRE-6A and IRE-7B, two ethyl-phenylcarbamate derivatives. Revista de Investigaci´on Cl´ınica 55, 444–447. Keister, D.B., 1983. Axenic culture of Giadia lamblia in TYI-S-33 medium supplemented with bile. Transactions of the Royal Society of Tropical Medicine and Hygiene 77, 487–488. Mart´ınez, S., Gonz´alez, J., Culebras, J., Mu˜no´ n, M., 2002. Los flavonoides: propiedades y acciones antioxidantes. Nutrici´on Hospitalaria 17, 271–278. Matsuda, H., Morikawa, T., Ando, S., Toguchida, I., Yoshikawa, M., 2003. Structural requirements of flavonoids for nitric oxide production inhibitory activity and mechanism of action. Bioorganic and Medicinal Chemistry 11, 1995–2000. Reynoldson, J., Thompson, R., Meloni, B., 1991. In vivo efficacy of albendazole against Giaria duodenalis in mice. Parasitology Research 77, 325–328. Roberts, I., Stevens, D., Mahmoud, A., Warren, K., 1976. Giardiasis in the mouse: an animal model. Gastroenterology 71, 57–61.