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Gastric antisecretory and antiulcer activities of an ethanolic extract of Bidens pilosa L. var. radiata Schult. Bip.
Journal of Ethnopharmacology 67 (1999) 333 – 340 www.elsevier.com/locate/jethpharm
Gastric antisecretory and antiulcer activities of an ethanolic extract of Bidens pilosa L. var. radiata Schult. Bip. A. Alvarez a, F. Pomar a, M.A. Sevilla b, M.J. Montero b,* a b
Departamento de In6estigaciones, Instituto de Gastroenterologı´a, Calle 25 No.503 entre H e I, Habana 10400, Cuba Laboratorio de Farmacognosia y Farmacodinamia, Departamento de Fisiologı´a y Farmacologı´a, Facultad de Farmacia, Uni6ersidad de Salamanca. Campus Miguel de Unamuno, 37007 Salamanca, Spain Received 9 September 1998; received in revised form 19 May 1999; accepted 24 May 1999
Abstract Bidens pilosa var. radiata Schult. Bip. is used in folk medicine to treat stomach disorders including peptic ulcers. The ethanolic extract (0.5–2 g/kg) decreased the gastric juice volume, acid secretion, as well as pepsin secretion in pylorus ligated rats. B. pilosa extract showed antiulcer activity against indomethacin-induced gastric lesions. The extract effectively inhibited gastric haemorragic lesions induced by ethanol, and with an effective dose of 2 g/kg being more potent than sucralfate (400 mg/kg). In contrast, ranitidine (50 mg/kg) failed to reduce these lesions. These results indicate that B. pilosa ethanolic extract exerts a cytoprotective effect in addition to its gastric antisecretory activity that could be due, partly at least, to the presence of flavonoids of which quercetin was identified by HPLC. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bidens pilosa L.; Compositae; Cytoprotection; Antiulcer; Antiacid
1. Introduction Bidens pilosa L. (Compositae) is originary from South America and can be found in almost all countries from tropical and subtropical regions. This plant is commonly used in the traditional medicine as a remedy for treating stomach disorders including peptic ulcer (Aces, 1939; SeoaneGallo, 1984). Pharmacognostic studies and phytochemical screenings of B. pilosa showed the presence of tannins, phytosterols, ascorbic acid, * Corresponding author. Tel.: +34-23-294530; fax: + 3423-294515.
carotene, essential oils, anthocianins, saponins, steroids and sugars (Doshchinskaya and Fetisova, 1975; Corral, 1986; Vasques et al., 1986). From this species, groups of compounds with biological activity, mainly, polyacetylenes and flavonoids have been isolated and identified. Isakova et al. (1986), in a phytochemical study covering a series of species of the genus Bidens, including B. pilosa, identified some types of flavonoids, mainly aurones and chalcones and Sashida et al. (1991) also isolated aurone glucosides from fresh leaves of B. pilosa. Several polyacetylenes have been isolated from the aerial parts and from the roots of B. pilosa (Geissberger and Se´quin, 1991; Sarg et al.,
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1991; Alvarez et al., 1996), the principal representative of this group of compounds being 1-phenylhepta-1,3,5-tryine. It seemed that the antimicrobial, antihelmintic and protozoocidal activities shown by different extracts of B. pilosa is due to its content of this polyacetylene (N’Dounga et al., 1983; Bondarenko et al., 1985; Geissberger and Se´quin, 1991). Other compounds with biological activities, such as terpenes, have also been isolated from this species (Geissberger and Se´quin, 1991; Zulueta et al., 1995). The aim of this study was to corroborate the antiulcer effect attributed to B. pilosa by evaluating the antiulcer activity of an ethanolic extract of a variety of this plant on indomethacin-, and ethanol-induced acute gastric lesions in rats. We also evaluated the effects on gastric secretion and mucosal production of mucus and nonprotein sulfhydryl groups, to explore possible ways of action of the extract.
kept in cages with raised mesh bottoms and deprived of solid diet 16 h before the onset of the experiment but received a nutritive solution of 8% sucrose in 0.2% NaCl to avoid excessive dehydration.
2. Materials and methods
2.4. Gastric secretion in pylorus-ligated rats
2.1. Plant material
The pylorus-ligated rat model described by Shay et al. (1945) was used. Under light ether anaesthesia, a small abdominal incision was made and the pylorus was ligated. Immediately B. pilosa extract (0.5, 1 and 2 g/kg), ranitidine (50 mg/kg), sucralfate (400 mg/kg) or vehicle (0.9% NaCl) were administered intraduodenally. Then the incision was closed and the animals were killed 4 h later. The stomach was excised, opened along the greatest curvature and the luminal contents were collected and centrifuged for 15 min at 4500 rpm to remove residual debris. The volume was measured and total acid ouput, determined by automatic titration, was expressed as microequivalents of H+ per 4 h. Pepsin concentration was determined by modification of the colorimetric method of Anson (1938) involving digestion of 2% haemoglobin in 0.02 N HCl (pH 2, 37°C, 15 min) followed by alcaline condensation with Folin-Ciocalteu reagent and spectrophotometric measurement at 578 nm. Pepsin output was expressed as mg of pepsin in 4h. Estimation of nonprotein sulphydryl groups (mg of glutation/g of stomach) and gastric mucus (ng of Alcian blue/ml/g of
The plant B. pilosa var. radiata Schult. Bip. was identified at the Experimental Station of Medicinal Plants from the Drug Development and Research Center, in Havana, Cuba. A voucher specimen (number 4598) was kept for record in the herbarium of the Experimental Station. The extract was prepared from dried ground aerial parts. A total of 1 kg of powdered crude drug was macerated for 48 h in 70% ethanol and was filtered then. The residue was 3-fold extracted with 70% ethanol for 1 h by mechanical stirring. The extracts were pooled, and concentrated under reduced pressure at 50°C until all the organic solvent was removed. Then it was lyophilized obtaining 165.2 g of ethanolic extract. Analysis by HPLC with UV detection at 245 nm showed that quercetin (0.09%) was present in the extract.
2.2. Animals The study was conducted on Wistar rats of either sex, weighing 160 – 200 g. The animals were
2.3. Extract and drug solutions, doses and treatments Lyophilized extract in doses of 0.5, 1 and 2 g/kg, and reference drugs ranitidine (Sigma) (50 mg/kg) and sucralfate (Merck Farma and Quı´mica, S.A.) (400 mg/kg) were reconstituted in tap water in case of experiments on gastroprotection, and in 0.9% saline in the study on gastric secretion. The extract and drug solutions were prepared freshly each time and administered intragastrically (gastroprotection) or intraduodenally (gastric secretion). The control rats received tap water or 0.9% saline in comparable volume (0.5 ml/100 g body weight) by the same routes.
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stomach) were done in accordance with the methods described by Sedlak and Lindsay (1968) and by Corne et al. (1974).
2.5. Indomethacin-induced gastric lesions The method of Lee et al. (1971) was followed. B. pilosa extract, ranitidine (doses as described above) and Vehicle (control group) were given orally 60 min before oral administration of indomethacin (20 mg/kg) in 4% sodium bicarbonate. The animals were sacrificed 5 h later, their stomachs removed, opened along the greatest curvature, rinsed under tap water and pinned on plastic polymer plates. Ulcer Index (U.I.) was obtained from the stereomicroscopic observation of the gastric lesions according to the score of Gamberini et al. (1991).
2.6. Ethanol-induced gastric lesions Lesions were induced according to Robert et al. (1979). B. pilosa extract, ranitidine, sucralfate (doses as described above), and vehicle (control group) were administered orally; 60 min later the rats were given 1 ml of 75% ethanol orally, and after another 60 min they were sacrificed and the stomachs removed. The remaining procedure was the same as described above for indomethacin-induced lesions. To determine if gastroprotective effect of the extract is exercised systematicaly or locally on the mucosa, its protective activity was assessed administering it intraperitoneally, in dose of 1 g/kg 1 h before the ethanol insult. To determine the influence of the B. pilosa extract on the effect of ethanol on gastric mucus secretion, a similar experiment to that of ethanol-induced lesions was carried out. The rats were pretreated with extracts at doses of 1 and 2 g/kg, ranitidine (50 mg/kg), or vehicle. Immediately after killing the animals the glandular segment of the stomach was removed, weighed and gastric mucus determined. In an experiment designed to determine whether mucosal protection by the extract is dependent on the synthesis of prostaglandins, rats were given indomethacin (10 mg/kg) subcutaneusly 60 min before administration of B. pilosa
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extract, at dose of 1 g/kg, or vehicle. In two other groups, rats were given 4% sodium bicarbonate (vehicle for indomethacin) before the extract or vehicle. All the animals received 1 ml of 75% ethanol orally 60 min after the extract administration and were killed 1 h later. Gastric protection was determined as described for experiments on ulcers induction.
2.7. Statistical analysis Data are presented as the mean9 SEM. The inhibitory ratio (%) was obtained by comparing the values in the treated animals with that of the control group. The significance of the differences between means was assessed using one-way ANOVA and Newman–Keuls tests. The Mann– Whitney test was applied to evaluate the differences between means in the experiment to determinate the influence of the extract on the effect of ethanol on gastric mucus. A P value of less than 0.05 was considered to be significant.
3. Results
3.1. Gastric secretion in pylorus-ligated rats Gastric secretion was evaluated as gastric juice volume, acid output, and pepsin output for 4 h after pylorus ligation. When administered immediately after ligation, B. pilosa extract (0.5, 1, 2 g/kg; intraduodenally) dose-dependently decreased the gastric juice volume, acid secretion, as well as pepsin secretion. This decrease reached statistical significance at doses of 1 and 2 g/kg for the volume, and at 2 g/kg for acid and pepsin secretions. Ranitidine (50 mg/kg) also caused a marked decrease in each secretory parameter, while sucralfate (400 mg/kg) did not show any modification on gastric secretion (Table 1). Mucus output did not show significant changes in response to any of the tested substances, except sucralfate (Table 2). In pylorus-ligated rats neither B. pilosa extract nor reference drugs modified the mucosal production of nonprotein sulfhydryl groups (data not shown).
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Table 1 Effect of ethanolic extract from B. pilosa L. var. radiata Schult. Bip, sucralfate and ranitidine on basal gastric secretion in pylorus-ligated ratsa Treatment
Vol. (ml/4 h)
Acid output (mEq H+/4 h
Pepsin output (mg/4 h)
Pepsin concentration (mg/ml)
Control (saline) Extract 0.5 g/kg Extract 1 g/kg Extract 2 g/kg Ranitidine 50 mg/kg Sucralfate 400 mg/kg
3.449 0.52 2.9590.62 1.869 0.32b 1.069 0.21b 1.7090.34b 2.929 0.48
282.399 56.42 273.24988.47 146.419 36.78 75.429 24.61b 54.719 23.34b 220.919 53.09
14.44 92.97 8.31 92.74 7.02 9 1.54 4.78 9 0.95b 3.17 9 1.46b 11.84 92.11
5.45 9 0.99 3.51 9 0.54 4.39 9 0.70 4.89 9 0.51 3.25 9 1.27 4.4490.95
a b
Each value represents the mean 9SEM of six animals. Significantly different from control PB0.05.
3.2. Indomethacin-induced gastric lesions Oral administration of indomethacin (20 mg/kg) resulted in production of gastric lesions predominantly on glandular segment of the stomach and few or none in the antrum. Lesions were produced linearly on mucosal folds and had appearance of mucosal erosions. In control rats an U.I. of 18.89 1.0 was observed, while B. pilosa extract significantly protected gastric mucosa against indomethacin-induced damage when administered at doses of 0.5 and 2 g/kg. Ranitidine showed the highest level of gastric protection (Table 3).
In order to determine whether the B. pilosa extract protected the gastric mucosa by a local or systemic effect, its cytoprotective activity against ethanol-induced gastric lesions was tested by intraperitoneal administration at a dose of 1 g/kg. The control value of U.I. was 11.89 0.8. In animals pretreated with the extract the ethanol-induced gastric damage was aggravated and the U.I. increased significantly to 15.59 1.1 (P B 0.05). Indomethacin treatment markedly increased about 75% the gastric ethanol-induced injury. The protective effect of the extract, however, was not abolished when prostaglandin biosynthesis had been inhibited beforehand by indomethacin. In
3.3. Ethanol-induced gastric lesions Oral administration of 75% ethanol produced severe haemorrhagic lesions in glandular mucosa consisting of elongated bands, usually parallel to the long axis of the stomach. As in indomethacin model, they were located mostly in the corpus. The control rats had an U.I. of 6.2 9 1.3. In animals pretreated with B. pilosa extract at doses of 1 and 2 g/kg, a significant inhibition of ethanol mucosal injury was detected, showing an U.I. of 1.49 0.8 and 1.090.4, respectively. The dose of 2 g/kg showed an inhibition of lesion formation (83.12%) greater than that of sucralfate (66.23%), which also significantly inhibited mucosal damage. By contrast, no reduction in ethanol-induced lesions was observed following the ranitidine administration (Table 4).
Table 2 Effect of ethanolic extract from B. pilosa L. var. radiata Schult. Bip, sucralfate and ranitidine on gastric mucus production in pylorus-ligated ratsa Treatment
N Mucus output (mg Alcian blue/ml per g tissue)
Control (saline) Ranitidine 50 mg/kg Sucralfate 400 mg/kg Extract 0.5 g/kg Extract 1 g/kg Extract 2 g/kg
7 6 6 6 7 7
a b
99.58 923.41 85.46 917.44 58.92 97.07b 89.96 9 10.15 67.15 9 9.06 103.63 9 17.14
Each value represents the mean 9SEM. Significantly different from control PB0.05.
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Table 3 Effect of ethanolic extract from B. pilosa L. var. radiata Schult. Bip and ranitidine on indomethacin-induced gastric lesionsa Treatment
N
Ulcer index
Inhibition (%)
Number of ulcers
Ulcerated animals
Control (vehicle) Ranitidine 50 mg/kg Extract 0.5 g/kg Extract 1 g/kg Extract 2 g/kg
7 8 5 5 6
18.829 1.02 9.759 1.20b 13.959 1.02b 16.80 92.02 12.049 1.17b
– 48.19 25.88 10.73 36.02
54.57 97.56 27.62 95.85b 43.0095.97 53.60 96.15 18.6792.15b
7/7 8/8 5/5 5/5 6/6
a b
Each value represents the mean 9SEM. Significantly different from control PB0.05.
groups which did not receive indomethacin the extract (1 g/kg) significantly decreased the U.I. produced by ethanol from 5.191.2 to 0.79 0.2 (P B0.01). In the indomethacin-pretreated groups the U.I. due to ethanol was 9.0 9 1.1 for vehicle control group and 3.190.6 (P B0.05) for extract treated group. In the experiment designed to investigate whether the extract influence the effect of ethanol on gastric mucus, we observed that ethanol significantly reduced mucus output (23.89 4.5) when compared to rats which received only vehicle (33.8 9 2.7). Oral pre-treatment with the extract dose-dependently protected against mucus depletion induced by ethanol, but the results reached statistical significance only at dose of 2 g/kg (40.0 9 8.2)
4. Discussion The findings of the present study demonstrate that B. pilosa L. var. radiata Schult. Bip possess gastric antisecretory, as well as mucosal protective activity. In pylorus-ligated rats, the intraduodenal administration of B. pilosa extract, at doses of 1 and 2 g/kg, produced a significant decrease of gastric juice volume; and at 2 g/kg significantly inhibited gastric acid and pepsin outputs. This inhibition was less than that of ranitidine (50 mg/kg). Sucralfate did not show any influence on gastric secretion as expected, since this drug exhibits both acute gastroprotection and chronic ulcer healing properties in patients and experimental animals without suppression of gastric acidity (Szabo and
Hollander, 1989). The reduced acid and pepsin outputs, measured after pylorus ligation, suggest that the protective mechanism of the extract on gastric mucosa might involve an inhibition of gastric secretion. A role for the gastric acid secretion in indomethacin-induced gastric injury was proposed by Satoh et al. (1981); they observed that administration of antisecretory drugs like atropine (2 mg/kg) and cimetidine (50 mg/kg) markedly inhibited the formation of indomethacin-induced gastric lesions. Arai et al. (1987) reported that indomethacin potentiated the stress-, 2-deoxy-Dglucose-, and vagal-stimulated gastric secretion, and produced severe gastric lesions. Later, these authors corroborate that indomethacin at doses within 1–30 mg/kg markedly increased both the stress-induced ulceration and the gastric acid secretion (Arai et al., 1993). These findings suggest that indomethacin-induced gastric lesions are related to a significant increase in the acidity of gastric juice. In the present study, the oral administration of B. pilosa extract, mainly at dose of 2 g/kg, significantly protected the gastric mucosa of rats against indomethacin-induced damage. This dose also showed to have a significant antisecretory activity. An antisecretory drug, ranitidine, also markedly inhibited the indomethacin-induced gastric lesions. These results suggest that the antiulcer activity of the extract against indomethacin injury might be related to its antisecretory effect. In the present study we also tested the B. pilosa extract against mucosal damage induced by ethanol, and observed that doses of 1 and 2 g/kg significantly inhibited the ethanol-induced gastric lesions. It is remarkable that these doses produced
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a greater protection than 400 mg/kg of sucralfate against the ethanol injury. The finding that ranitidine orally did not protect the gastric mucosa against ethanol-induced damage is in agreement with the works of other authors (Del Soldato et al., 1985; Hakkinen et al., 1991; Palacios et al., 1995, 1998). The results of antiulcer and antisecretory studies showed that the extract was active after either oral or intraduodenal administration. Moreover, the fact that the extract protected against ethanolinduced lesions at a lower dose than that required to significantly reduce the acid and pepsin output, suggests that a cytoprotective mechanism may be involved. The genesis of ethanol-induced gastric lesions is of multifactorial origin with the decrease in gastric mucus amount as one of the involved factors. A depletion of stomach wall mucus after oral ethanol administration has been reported (AlHarbi et al., 1997). In the present study we corroborate that finding and observed that at dose of 2 g/kg, the extract not only significantly inhibited the gastric mucus depletion produced by ethanol, but also the mucus output was greater than that observed in stomachs of control. This fact might be related to the recovery of gastric mucus induced by the extract. Ranitidine did not overcome the depletion of mucus induced by ethanol, which was expected since its antiulcerogenic efficacy is due mainly to its antagonism on histaminergic activity as a consequence of the H2-receptors blockade.
In a previous work, in rats pretreated with an ethanolic extract of B. pilosa (1.5 g/kg) before cold-restraint stress, we observed by histochemical assessment that the extract markedly inhibited stress-induced mucosal depletion of neutral glycoproteins, and the mucosal content of neutral glycoproteins in rats pretreated with the extract was similar to that observed in non-stressed control animals (Alvarez et al., 1990). In a study on the antiulcer activity of another species of the genus Bidens, Alarco´n de la Lastra et al. (1994a) obtained similar results. These facts suggest that the ability of B. pilosa extract to inhibit the gastric mucus depletion, as observed in our present study, could be responsable of the increased mucosal resistance against the ethanol-induced damage. The intraduodenal administration of the extract in pylorus-ligated rats did not produce significant changes in mucus production, suggesting that the effect on gastric mucus depends on contact of the extract with the gastric mucosa. When the extract was administered intraperitoneally at a dose of 1 g/kg, it did not protect, but even aggravated the ethanol-induced damage. This fact can be explained because i.p. injection of a crude extract, which contain non-physiological substances (abundant in crude plant extracts), might produce peritoneal irritation in rats and a direct toxic action as expected from crude materials. The fact that the enhancing action on mucus production was only present when the extract was administered orally, supports the hypothesis that the gastroprotective effect of the extract is dependent mainly on the contact with the gastric mucosa.
Table 4 Effect of ethanolic extract from B. pilosa L. var. radiata Schult. Bip, sucralfate and ranitidine on ethanol-induced gastric haemorrhagic lesions in the ratsa Treatment
N
Ulcer index
Inhibition (%)
Number of ulcers
Ulcerated animals
Control (vehicle) Ranitidine 50 mg/kg Sucralfate 400 mg/kg Extract 0.5 g/kg Extract 1 g/kg Extract 2 g/kg
11 10 12 6 6 6
6.169 1.34 5.979 1.34 2.08 9 0.45b 6.469 1.55 1.37 9 0.84b 1.0490.42b
– 3.08 66.23 −4.87 77.60 83.12
5.00 9 1.83 5.10 91.21 0.83 90.24b 4.67 91.96 0.67 9 0.67 0.17 9 0.17
7/11 9/10 7/12 5/6 1/6 1/6
a b
Each value represents the mean 9SEM. Significantly different from control PB0.05.
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Pretreatment with indomethacin alone increased the injury after ethanol gavage. Nevertheless, the gastroprotective effect of the extract on gastric mucosa remained despite the indomethacin pretreatment, indicating that the presence of endogenous prostaglandins is not essential to the expression of mucosal protective activity of the extract. This fact supports the suggestion that gastroprotection exerted by the extract might be related, mainly, to the stimulation of mucus secretion, thus streghthening the gastric mucus-bicarbonate barrier. In this study we found that mucosal NP-SH production seemed not to be modified by the extract when it is administered intraduodenally. It is known that different compounds from plant origin, such as terpenes, anthraquinones and flavonoids, have shown inhibitory effect on gastric acid secretion and/or antiulcer activity against several experimental models (Pillai and Santhakumari, 1984; Murakami et al., 1992). In an attempt to correlate the effects of the extract observed in this study with chemical compounds present in B. pilosa, we carried out a literature review and found that flavonoids present in many plant species have a broad scale of biological activities, mainly, antiinflammatory and antiulcer (Yamahara et al., 1990; Murakami et al., 1990; Alarco´n de la Lastra et al., 1993). Quercetin is a flavonoid which has been isolated from B. pilosa (Geissberger and Se´quin, 1991) and of which antiulcer and antisecretory effects have been reported (Alarco´n de la Lastra et al., 1994b). These data suggest that flavonoids present in B. pilosa might be active principles responsible, partly at least, of the antiulcer and antisecretory activities shown by the tested extract. In conclusion, it appears that B. pilosa var. radiata Schult. Bip possess antiulcerogenic principles which protect against gastric mucosal damage induced by indomethacin and ethanol, through inhibition of gastric acid and pepsin output (attenuation of aggresive factors) and stimulation of mucus secretion (potentiation of defensive factors). Probably the antiulcer effect is due, partly at least, to the presence of flavonoids in the ethanolic extract since quercetin has been identified by HPLC, although the involvement of
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other compounds in the plant cannot be ruled out. The data so far obtained do not indicate, however, which specific mechanism(s) is (are) responsible for the antisecretory and antiulcer activities. Further studies are required to isolate the antiulcer compounds and to elucidate their mechanisms of action.
Acknowledgements The authors thank Dr Pilar Go´mez-Serranillos of Universidad Complutense, Madrid for HPLC analysis.
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and antiparasitic activity of 1-phenyl-1,3,5-heptatriyne. Plantes Medicinales et Phytotherapie 17, 64 – 75. Palacios, B., Montero, M.J., Sevilla, M.A., San Roma´n, L., 1995. JB-9322, a new selective histamine H2-receptor antagonist with potent gastric mucosal protective properties. British Journal of Pharmacology 115, 57 – 66. Palacios, B., Montero, M.J., Sevilla, M.A., San Roma´n, L., 1998. Pharmacology of JB-9315. A new selective histamine H2-receptor antagonist. General Pharmacology 30 (2), 181 – 189. Pillai, N.R., Santhakumari, G., 1984. Effects of nimbidin on acute and chronic gastro-duodenal ulcer models in experimental animals. Planta Medica 50, 143 – 146. Robert, A., Nezamis, J., Lancaster, C., Hanchar, A., 1979. Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaOH and thermal injury. Gastroenterology 77, 433 – 443. Sarg, T.M., Ateya, A.M., Farrag, N.M., Abbas, F.A., 1991. Constituents and biological activity of Bidens pilosa L. grown in Egypt. Acta Pharmaceutica Hungarica 61, 317 – 323. Sashida, Y., Ogawa, K., Kitada, M., Karikome, H., Mimaki, Y., Shimomura, H., 1991. New aurone glucoside and new phenylpropanid glucosides from Bidens pilosa. Chemical and Pharmaceutical Bulletin 39, 709 – 711. Satoh, H., Inada, I., Hirata, T., Maki, Y., 1981. Indomethacin produces gastric antral ulcers in the refed rat. Gastroenterology 81, 719 – 725. Sedlak, J., Lindsay, R.H., 1968. Estimation of total proteinbound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Analytical Biochemistry 25, 192 – 205. Seoane-Gallo, J., 1984. El Folclor Me´dico de Cuba. Editora Ciencias Sociales, La Habana, p. 770. Shay, H., Komarov, S.A., Fels, S.S., Meranze, D., Gruenstein, M., Siplet, H., 1945. A simple method for the uniform production of gastric ulceration in rat. Gastroenterology 5, 43 – 61. Szabo, S., Hollander, D., 1989. Pathways of gastrointestinal protection and repair: mechanisms of action of sucralfate. American Journal of Medicine 86 (Suppl. 6A), 23 – 31. Vasques, C.A.V., Vasques, N.V., Geller, M., 1986. Contribucao ao estudo do picao (Bidens pilosa L.). Arquivos Brasileiros de Medicina 60, 283 – 287. Yamahara, J., Mochizuki, M., Fujimura, H., Takaishi, Y., Yoshida, M., Tomimatsu, T., Tamai, Y., 1990. Antiulcer action of Sophora fla6escens root and an active constituent. I. Journal of Ethnopharmacology 29, 173 – 177. Zulueta, C.A., Zulueta, A., Tada, M., Ragasa, C.Y., 1995. A diterpene from Bidens pilosa. Phytochemistry 38, 1449 – 1450.
Gastric antisecretory and antiulcer activities of an ethanolic extract of Bidens pilosa L. var. radiata Schult. Bip. A. Alvarez a, F. Pomar a, M.A. Sevilla b, M.J. Montero b,* a b
Departamento de In6estigaciones, Instituto de Gastroenterologı´a, Calle 25 No.503 entre H e I, Habana 10400, Cuba Laboratorio de Farmacognosia y Farmacodinamia, Departamento de Fisiologı´a y Farmacologı´a, Facultad de Farmacia, Uni6ersidad de Salamanca. Campus Miguel de Unamuno, 37007 Salamanca, Spain Received 9 September 1998; received in revised form 19 May 1999; accepted 24 May 1999
Abstract Bidens pilosa var. radiata Schult. Bip. is used in folk medicine to treat stomach disorders including peptic ulcers. The ethanolic extract (0.5–2 g/kg) decreased the gastric juice volume, acid secretion, as well as pepsin secretion in pylorus ligated rats. B. pilosa extract showed antiulcer activity against indomethacin-induced gastric lesions. The extract effectively inhibited gastric haemorragic lesions induced by ethanol, and with an effective dose of 2 g/kg being more potent than sucralfate (400 mg/kg). In contrast, ranitidine (50 mg/kg) failed to reduce these lesions. These results indicate that B. pilosa ethanolic extract exerts a cytoprotective effect in addition to its gastric antisecretory activity that could be due, partly at least, to the presence of flavonoids of which quercetin was identified by HPLC. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bidens pilosa L.; Compositae; Cytoprotection; Antiulcer; Antiacid
1. Introduction Bidens pilosa L. (Compositae) is originary from South America and can be found in almost all countries from tropical and subtropical regions. This plant is commonly used in the traditional medicine as a remedy for treating stomach disorders including peptic ulcer (Aces, 1939; SeoaneGallo, 1984). Pharmacognostic studies and phytochemical screenings of B. pilosa showed the presence of tannins, phytosterols, ascorbic acid, * Corresponding author. Tel.: +34-23-294530; fax: + 3423-294515.
carotene, essential oils, anthocianins, saponins, steroids and sugars (Doshchinskaya and Fetisova, 1975; Corral, 1986; Vasques et al., 1986). From this species, groups of compounds with biological activity, mainly, polyacetylenes and flavonoids have been isolated and identified. Isakova et al. (1986), in a phytochemical study covering a series of species of the genus Bidens, including B. pilosa, identified some types of flavonoids, mainly aurones and chalcones and Sashida et al. (1991) also isolated aurone glucosides from fresh leaves of B. pilosa. Several polyacetylenes have been isolated from the aerial parts and from the roots of B. pilosa (Geissberger and Se´quin, 1991; Sarg et al.,
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1991; Alvarez et al., 1996), the principal representative of this group of compounds being 1-phenylhepta-1,3,5-tryine. It seemed that the antimicrobial, antihelmintic and protozoocidal activities shown by different extracts of B. pilosa is due to its content of this polyacetylene (N’Dounga et al., 1983; Bondarenko et al., 1985; Geissberger and Se´quin, 1991). Other compounds with biological activities, such as terpenes, have also been isolated from this species (Geissberger and Se´quin, 1991; Zulueta et al., 1995). The aim of this study was to corroborate the antiulcer effect attributed to B. pilosa by evaluating the antiulcer activity of an ethanolic extract of a variety of this plant on indomethacin-, and ethanol-induced acute gastric lesions in rats. We also evaluated the effects on gastric secretion and mucosal production of mucus and nonprotein sulfhydryl groups, to explore possible ways of action of the extract.
kept in cages with raised mesh bottoms and deprived of solid diet 16 h before the onset of the experiment but received a nutritive solution of 8% sucrose in 0.2% NaCl to avoid excessive dehydration.
2. Materials and methods
2.4. Gastric secretion in pylorus-ligated rats
2.1. Plant material
The pylorus-ligated rat model described by Shay et al. (1945) was used. Under light ether anaesthesia, a small abdominal incision was made and the pylorus was ligated. Immediately B. pilosa extract (0.5, 1 and 2 g/kg), ranitidine (50 mg/kg), sucralfate (400 mg/kg) or vehicle (0.9% NaCl) were administered intraduodenally. Then the incision was closed and the animals were killed 4 h later. The stomach was excised, opened along the greatest curvature and the luminal contents were collected and centrifuged for 15 min at 4500 rpm to remove residual debris. The volume was measured and total acid ouput, determined by automatic titration, was expressed as microequivalents of H+ per 4 h. Pepsin concentration was determined by modification of the colorimetric method of Anson (1938) involving digestion of 2% haemoglobin in 0.02 N HCl (pH 2, 37°C, 15 min) followed by alcaline condensation with Folin-Ciocalteu reagent and spectrophotometric measurement at 578 nm. Pepsin output was expressed as mg of pepsin in 4h. Estimation of nonprotein sulphydryl groups (mg of glutation/g of stomach) and gastric mucus (ng of Alcian blue/ml/g of
The plant B. pilosa var. radiata Schult. Bip. was identified at the Experimental Station of Medicinal Plants from the Drug Development and Research Center, in Havana, Cuba. A voucher specimen (number 4598) was kept for record in the herbarium of the Experimental Station. The extract was prepared from dried ground aerial parts. A total of 1 kg of powdered crude drug was macerated for 48 h in 70% ethanol and was filtered then. The residue was 3-fold extracted with 70% ethanol for 1 h by mechanical stirring. The extracts were pooled, and concentrated under reduced pressure at 50°C until all the organic solvent was removed. Then it was lyophilized obtaining 165.2 g of ethanolic extract. Analysis by HPLC with UV detection at 245 nm showed that quercetin (0.09%) was present in the extract.
2.2. Animals The study was conducted on Wistar rats of either sex, weighing 160 – 200 g. The animals were
2.3. Extract and drug solutions, doses and treatments Lyophilized extract in doses of 0.5, 1 and 2 g/kg, and reference drugs ranitidine (Sigma) (50 mg/kg) and sucralfate (Merck Farma and Quı´mica, S.A.) (400 mg/kg) were reconstituted in tap water in case of experiments on gastroprotection, and in 0.9% saline in the study on gastric secretion. The extract and drug solutions were prepared freshly each time and administered intragastrically (gastroprotection) or intraduodenally (gastric secretion). The control rats received tap water or 0.9% saline in comparable volume (0.5 ml/100 g body weight) by the same routes.
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stomach) were done in accordance with the methods described by Sedlak and Lindsay (1968) and by Corne et al. (1974).
2.5. Indomethacin-induced gastric lesions The method of Lee et al. (1971) was followed. B. pilosa extract, ranitidine (doses as described above) and Vehicle (control group) were given orally 60 min before oral administration of indomethacin (20 mg/kg) in 4% sodium bicarbonate. The animals were sacrificed 5 h later, their stomachs removed, opened along the greatest curvature, rinsed under tap water and pinned on plastic polymer plates. Ulcer Index (U.I.) was obtained from the stereomicroscopic observation of the gastric lesions according to the score of Gamberini et al. (1991).
2.6. Ethanol-induced gastric lesions Lesions were induced according to Robert et al. (1979). B. pilosa extract, ranitidine, sucralfate (doses as described above), and vehicle (control group) were administered orally; 60 min later the rats were given 1 ml of 75% ethanol orally, and after another 60 min they were sacrificed and the stomachs removed. The remaining procedure was the same as described above for indomethacin-induced lesions. To determine if gastroprotective effect of the extract is exercised systematicaly or locally on the mucosa, its protective activity was assessed administering it intraperitoneally, in dose of 1 g/kg 1 h before the ethanol insult. To determine the influence of the B. pilosa extract on the effect of ethanol on gastric mucus secretion, a similar experiment to that of ethanol-induced lesions was carried out. The rats were pretreated with extracts at doses of 1 and 2 g/kg, ranitidine (50 mg/kg), or vehicle. Immediately after killing the animals the glandular segment of the stomach was removed, weighed and gastric mucus determined. In an experiment designed to determine whether mucosal protection by the extract is dependent on the synthesis of prostaglandins, rats were given indomethacin (10 mg/kg) subcutaneusly 60 min before administration of B. pilosa
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extract, at dose of 1 g/kg, or vehicle. In two other groups, rats were given 4% sodium bicarbonate (vehicle for indomethacin) before the extract or vehicle. All the animals received 1 ml of 75% ethanol orally 60 min after the extract administration and were killed 1 h later. Gastric protection was determined as described for experiments on ulcers induction.
2.7. Statistical analysis Data are presented as the mean9 SEM. The inhibitory ratio (%) was obtained by comparing the values in the treated animals with that of the control group. The significance of the differences between means was assessed using one-way ANOVA and Newman–Keuls tests. The Mann– Whitney test was applied to evaluate the differences between means in the experiment to determinate the influence of the extract on the effect of ethanol on gastric mucus. A P value of less than 0.05 was considered to be significant.
3. Results
3.1. Gastric secretion in pylorus-ligated rats Gastric secretion was evaluated as gastric juice volume, acid output, and pepsin output for 4 h after pylorus ligation. When administered immediately after ligation, B. pilosa extract (0.5, 1, 2 g/kg; intraduodenally) dose-dependently decreased the gastric juice volume, acid secretion, as well as pepsin secretion. This decrease reached statistical significance at doses of 1 and 2 g/kg for the volume, and at 2 g/kg for acid and pepsin secretions. Ranitidine (50 mg/kg) also caused a marked decrease in each secretory parameter, while sucralfate (400 mg/kg) did not show any modification on gastric secretion (Table 1). Mucus output did not show significant changes in response to any of the tested substances, except sucralfate (Table 2). In pylorus-ligated rats neither B. pilosa extract nor reference drugs modified the mucosal production of nonprotein sulfhydryl groups (data not shown).
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Table 1 Effect of ethanolic extract from B. pilosa L. var. radiata Schult. Bip, sucralfate and ranitidine on basal gastric secretion in pylorus-ligated ratsa Treatment
Vol. (ml/4 h)
Acid output (mEq H+/4 h
Pepsin output (mg/4 h)
Pepsin concentration (mg/ml)
Control (saline) Extract 0.5 g/kg Extract 1 g/kg Extract 2 g/kg Ranitidine 50 mg/kg Sucralfate 400 mg/kg
3.449 0.52 2.9590.62 1.869 0.32b 1.069 0.21b 1.7090.34b 2.929 0.48
282.399 56.42 273.24988.47 146.419 36.78 75.429 24.61b 54.719 23.34b 220.919 53.09
14.44 92.97 8.31 92.74 7.02 9 1.54 4.78 9 0.95b 3.17 9 1.46b 11.84 92.11
5.45 9 0.99 3.51 9 0.54 4.39 9 0.70 4.89 9 0.51 3.25 9 1.27 4.4490.95
a b
Each value represents the mean 9SEM of six animals. Significantly different from control PB0.05.
3.2. Indomethacin-induced gastric lesions Oral administration of indomethacin (20 mg/kg) resulted in production of gastric lesions predominantly on glandular segment of the stomach and few or none in the antrum. Lesions were produced linearly on mucosal folds and had appearance of mucosal erosions. In control rats an U.I. of 18.89 1.0 was observed, while B. pilosa extract significantly protected gastric mucosa against indomethacin-induced damage when administered at doses of 0.5 and 2 g/kg. Ranitidine showed the highest level of gastric protection (Table 3).
In order to determine whether the B. pilosa extract protected the gastric mucosa by a local or systemic effect, its cytoprotective activity against ethanol-induced gastric lesions was tested by intraperitoneal administration at a dose of 1 g/kg. The control value of U.I. was 11.89 0.8. In animals pretreated with the extract the ethanol-induced gastric damage was aggravated and the U.I. increased significantly to 15.59 1.1 (P B 0.05). Indomethacin treatment markedly increased about 75% the gastric ethanol-induced injury. The protective effect of the extract, however, was not abolished when prostaglandin biosynthesis had been inhibited beforehand by indomethacin. In
3.3. Ethanol-induced gastric lesions Oral administration of 75% ethanol produced severe haemorrhagic lesions in glandular mucosa consisting of elongated bands, usually parallel to the long axis of the stomach. As in indomethacin model, they were located mostly in the corpus. The control rats had an U.I. of 6.2 9 1.3. In animals pretreated with B. pilosa extract at doses of 1 and 2 g/kg, a significant inhibition of ethanol mucosal injury was detected, showing an U.I. of 1.49 0.8 and 1.090.4, respectively. The dose of 2 g/kg showed an inhibition of lesion formation (83.12%) greater than that of sucralfate (66.23%), which also significantly inhibited mucosal damage. By contrast, no reduction in ethanol-induced lesions was observed following the ranitidine administration (Table 4).
Table 2 Effect of ethanolic extract from B. pilosa L. var. radiata Schult. Bip, sucralfate and ranitidine on gastric mucus production in pylorus-ligated ratsa Treatment
N Mucus output (mg Alcian blue/ml per g tissue)
Control (saline) Ranitidine 50 mg/kg Sucralfate 400 mg/kg Extract 0.5 g/kg Extract 1 g/kg Extract 2 g/kg
7 6 6 6 7 7
a b
99.58 923.41 85.46 917.44 58.92 97.07b 89.96 9 10.15 67.15 9 9.06 103.63 9 17.14
Each value represents the mean 9SEM. Significantly different from control PB0.05.
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Table 3 Effect of ethanolic extract from B. pilosa L. var. radiata Schult. Bip and ranitidine on indomethacin-induced gastric lesionsa Treatment
N
Ulcer index
Inhibition (%)
Number of ulcers
Ulcerated animals
Control (vehicle) Ranitidine 50 mg/kg Extract 0.5 g/kg Extract 1 g/kg Extract 2 g/kg
7 8 5 5 6
18.829 1.02 9.759 1.20b 13.959 1.02b 16.80 92.02 12.049 1.17b
– 48.19 25.88 10.73 36.02
54.57 97.56 27.62 95.85b 43.0095.97 53.60 96.15 18.6792.15b
7/7 8/8 5/5 5/5 6/6
a b
Each value represents the mean 9SEM. Significantly different from control PB0.05.
groups which did not receive indomethacin the extract (1 g/kg) significantly decreased the U.I. produced by ethanol from 5.191.2 to 0.79 0.2 (P B0.01). In the indomethacin-pretreated groups the U.I. due to ethanol was 9.0 9 1.1 for vehicle control group and 3.190.6 (P B0.05) for extract treated group. In the experiment designed to investigate whether the extract influence the effect of ethanol on gastric mucus, we observed that ethanol significantly reduced mucus output (23.89 4.5) when compared to rats which received only vehicle (33.8 9 2.7). Oral pre-treatment with the extract dose-dependently protected against mucus depletion induced by ethanol, but the results reached statistical significance only at dose of 2 g/kg (40.0 9 8.2)
4. Discussion The findings of the present study demonstrate that B. pilosa L. var. radiata Schult. Bip possess gastric antisecretory, as well as mucosal protective activity. In pylorus-ligated rats, the intraduodenal administration of B. pilosa extract, at doses of 1 and 2 g/kg, produced a significant decrease of gastric juice volume; and at 2 g/kg significantly inhibited gastric acid and pepsin outputs. This inhibition was less than that of ranitidine (50 mg/kg). Sucralfate did not show any influence on gastric secretion as expected, since this drug exhibits both acute gastroprotection and chronic ulcer healing properties in patients and experimental animals without suppression of gastric acidity (Szabo and
Hollander, 1989). The reduced acid and pepsin outputs, measured after pylorus ligation, suggest that the protective mechanism of the extract on gastric mucosa might involve an inhibition of gastric secretion. A role for the gastric acid secretion in indomethacin-induced gastric injury was proposed by Satoh et al. (1981); they observed that administration of antisecretory drugs like atropine (2 mg/kg) and cimetidine (50 mg/kg) markedly inhibited the formation of indomethacin-induced gastric lesions. Arai et al. (1987) reported that indomethacin potentiated the stress-, 2-deoxy-Dglucose-, and vagal-stimulated gastric secretion, and produced severe gastric lesions. Later, these authors corroborate that indomethacin at doses within 1–30 mg/kg markedly increased both the stress-induced ulceration and the gastric acid secretion (Arai et al., 1993). These findings suggest that indomethacin-induced gastric lesions are related to a significant increase in the acidity of gastric juice. In the present study, the oral administration of B. pilosa extract, mainly at dose of 2 g/kg, significantly protected the gastric mucosa of rats against indomethacin-induced damage. This dose also showed to have a significant antisecretory activity. An antisecretory drug, ranitidine, also markedly inhibited the indomethacin-induced gastric lesions. These results suggest that the antiulcer activity of the extract against indomethacin injury might be related to its antisecretory effect. In the present study we also tested the B. pilosa extract against mucosal damage induced by ethanol, and observed that doses of 1 and 2 g/kg significantly inhibited the ethanol-induced gastric lesions. It is remarkable that these doses produced
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a greater protection than 400 mg/kg of sucralfate against the ethanol injury. The finding that ranitidine orally did not protect the gastric mucosa against ethanol-induced damage is in agreement with the works of other authors (Del Soldato et al., 1985; Hakkinen et al., 1991; Palacios et al., 1995, 1998). The results of antiulcer and antisecretory studies showed that the extract was active after either oral or intraduodenal administration. Moreover, the fact that the extract protected against ethanolinduced lesions at a lower dose than that required to significantly reduce the acid and pepsin output, suggests that a cytoprotective mechanism may be involved. The genesis of ethanol-induced gastric lesions is of multifactorial origin with the decrease in gastric mucus amount as one of the involved factors. A depletion of stomach wall mucus after oral ethanol administration has been reported (AlHarbi et al., 1997). In the present study we corroborate that finding and observed that at dose of 2 g/kg, the extract not only significantly inhibited the gastric mucus depletion produced by ethanol, but also the mucus output was greater than that observed in stomachs of control. This fact might be related to the recovery of gastric mucus induced by the extract. Ranitidine did not overcome the depletion of mucus induced by ethanol, which was expected since its antiulcerogenic efficacy is due mainly to its antagonism on histaminergic activity as a consequence of the H2-receptors blockade.
In a previous work, in rats pretreated with an ethanolic extract of B. pilosa (1.5 g/kg) before cold-restraint stress, we observed by histochemical assessment that the extract markedly inhibited stress-induced mucosal depletion of neutral glycoproteins, and the mucosal content of neutral glycoproteins in rats pretreated with the extract was similar to that observed in non-stressed control animals (Alvarez et al., 1990). In a study on the antiulcer activity of another species of the genus Bidens, Alarco´n de la Lastra et al. (1994a) obtained similar results. These facts suggest that the ability of B. pilosa extract to inhibit the gastric mucus depletion, as observed in our present study, could be responsable of the increased mucosal resistance against the ethanol-induced damage. The intraduodenal administration of the extract in pylorus-ligated rats did not produce significant changes in mucus production, suggesting that the effect on gastric mucus depends on contact of the extract with the gastric mucosa. When the extract was administered intraperitoneally at a dose of 1 g/kg, it did not protect, but even aggravated the ethanol-induced damage. This fact can be explained because i.p. injection of a crude extract, which contain non-physiological substances (abundant in crude plant extracts), might produce peritoneal irritation in rats and a direct toxic action as expected from crude materials. The fact that the enhancing action on mucus production was only present when the extract was administered orally, supports the hypothesis that the gastroprotective effect of the extract is dependent mainly on the contact with the gastric mucosa.
Table 4 Effect of ethanolic extract from B. pilosa L. var. radiata Schult. Bip, sucralfate and ranitidine on ethanol-induced gastric haemorrhagic lesions in the ratsa Treatment
N
Ulcer index
Inhibition (%)
Number of ulcers
Ulcerated animals
Control (vehicle) Ranitidine 50 mg/kg Sucralfate 400 mg/kg Extract 0.5 g/kg Extract 1 g/kg Extract 2 g/kg
11 10 12 6 6 6
6.169 1.34 5.979 1.34 2.08 9 0.45b 6.469 1.55 1.37 9 0.84b 1.0490.42b
– 3.08 66.23 −4.87 77.60 83.12
5.00 9 1.83 5.10 91.21 0.83 90.24b 4.67 91.96 0.67 9 0.67 0.17 9 0.17
7/11 9/10 7/12 5/6 1/6 1/6
a b
Each value represents the mean 9SEM. Significantly different from control PB0.05.
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Pretreatment with indomethacin alone increased the injury after ethanol gavage. Nevertheless, the gastroprotective effect of the extract on gastric mucosa remained despite the indomethacin pretreatment, indicating that the presence of endogenous prostaglandins is not essential to the expression of mucosal protective activity of the extract. This fact supports the suggestion that gastroprotection exerted by the extract might be related, mainly, to the stimulation of mucus secretion, thus streghthening the gastric mucus-bicarbonate barrier. In this study we found that mucosal NP-SH production seemed not to be modified by the extract when it is administered intraduodenally. It is known that different compounds from plant origin, such as terpenes, anthraquinones and flavonoids, have shown inhibitory effect on gastric acid secretion and/or antiulcer activity against several experimental models (Pillai and Santhakumari, 1984; Murakami et al., 1992). In an attempt to correlate the effects of the extract observed in this study with chemical compounds present in B. pilosa, we carried out a literature review and found that flavonoids present in many plant species have a broad scale of biological activities, mainly, antiinflammatory and antiulcer (Yamahara et al., 1990; Murakami et al., 1990; Alarco´n de la Lastra et al., 1993). Quercetin is a flavonoid which has been isolated from B. pilosa (Geissberger and Se´quin, 1991) and of which antiulcer and antisecretory effects have been reported (Alarco´n de la Lastra et al., 1994b). These data suggest that flavonoids present in B. pilosa might be active principles responsible, partly at least, of the antiulcer and antisecretory activities shown by the tested extract. In conclusion, it appears that B. pilosa var. radiata Schult. Bip possess antiulcerogenic principles which protect against gastric mucosal damage induced by indomethacin and ethanol, through inhibition of gastric acid and pepsin output (attenuation of aggresive factors) and stimulation of mucus secretion (potentiation of defensive factors). Probably the antiulcer effect is due, partly at least, to the presence of flavonoids in the ethanolic extract since quercetin has been identified by HPLC, although the involvement of
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other compounds in the plant cannot be ruled out. The data so far obtained do not indicate, however, which specific mechanism(s) is (are) responsible for the antisecretory and antiulcer activities. Further studies are required to isolate the antiulcer compounds and to elucidate their mechanisms of action.
Acknowledgements The authors thank Dr Pilar Go´mez-Serranillos of Universidad Complutense, Madrid for HPLC analysis.
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