- Email: [email protected]
Anti-ulcer actions of the bark methanol extract of Voacanga africana in different experimental ulcer models in rats
Journal of Ethnopharmacology 73 (2000) 423 – 428 www.elsevier.com/locate/jethpharm
Anti-ulcer actions of the bark methanol extract of Voacanga africana in different experimental ulcer models in rats Paul V. Tan a,*, Veronique B. Penlap b, Barthelemy Nyasse c, Joseph D.B. Nguemo b a
Department of Animal Biology and Physiology, Faculty of Science, Uni6ersity of Yaounde I, P.O. Box 812, Yaounde, Cameroon b Department of Biochemistry, Faculty of Science, Uni6ersity of Yaounde I, P.O. Box 812, Yaounde, Cameroon c Department of Organic Chemistry, Faculty of Science, Uni6ersity of Yaounde I, P.O. Box 812, Yaounde, Cameroon Received 26 January 2000; received in revised form 28 May 2000; accepted 6 June 2000
Abstract The antiulcerogenic effects of the bark methanol extract of Voacanga africana were studied using various experimental ulcer models in rats. The effects of the extract on the volume of gastric juice, gastric pH, acid output, mucus production and peptic activity were recorded, as well as the preventive action against lesions caused by HCl/ethanol and indomethacin. Oral administration of the extract (500 – 750 mg/kg) inhibited the formation of gastric lesions induced by HCl/ethanol (40–63% inhibition). The inhibitory effect against HCl/ethanol was significantly (PB 0.01) suppressed by pre-treatment of the rats with indomethacin (30 mg/kg, i.p.). The extract significantly reduced gastric lesion formation in pylorus ligated rats, but this was not associated with an increase in gastric mucus production or with a reduction in acid content, volume of gastric secretion or pepsin activity of the gastric juice. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Voacanga africana; Apocynaceae; Bark methanol extract; Anti-ulcer activity
1. Introduction Voacanga africana Staph. (Apocynaceae) is a tree plant occurring in all of West Africa and as far as the Congo and even Tanzania. In Cote d’Ivoire, leprosy, diarrhoea, generalized oedema, convulsions in children and madness figure among the ethno-medical applications of the plant (Bouquet and Debray, 1974). In Cameroon, the fruit, bark and leaf extracts are used to treat cases of * Corresponding author.
orchitis, ectopic testes and gonorrhoea, respectively (Adjanohoun et al., 1996). Information provided by practitioners of traditional medicine suggest that V. africana also possesses useful antiulcer properties, although this was not cited in the national ethno-botanical survey of Cameroonian medicinal plants conducted by Adjanohoun et al. (1996). In a previous study, the gastric protective effect of the bark aqueous extract of V. africana against HCl/ethanol solution was demonstrated (Tan et al., 1997a). When further tested in the pylorus
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 2 9 2 - 0
424
P.V. Tan et al. / Journal of Ethnopharmacology 73 (2000) 423–428
ligated rat model, the extract prevented gastric mucosal damage, but did not significantly reduce the acidity of gastric juice (Tan et al., 1997b). However, we have observed in previous work (Tan et al., 1996) that gastric acidity within the 50 – 60 mEq/l range causes severe gastric mucosal ulceration. This ulcerative effect of the gastric juice is due to the presence of pepsin and HCl that auto digest the gastric mucosa (Shay et al., 1945). The present paper describes the effects of the bark methanol extract of V. africana in different experimental ulcer models. The possible mode of action of the extract against the pylorus ligated ulcer model was investigated and discussed.
of saline orally. Another group of rats received 100 mg/kg of commercial sucralfate (Usiphar-F; Compeigne, France). The HCl/ethanol solution (150 mM HCl in 60% v/v ethanol) was given 1 h later. The rats were killed after 1 h using ether. The stomachs were removed and cut open along the greater curvature. The length and width of each lesion in the glandular portion was measured and the lesions were scored as described previously (Tan et al., 1996). Percentage ulcerated surface was calculated by expressing the total area (mm2) covered by the lesions as a proportion of the total corpus mucosal surface.
2. Materials and methods
2.4. HCl/ethanol-induced ulcers in rats pre-treated with indomethacin
2.1. Animals Three-month old male albino Wistar rats (160 – 200 g) were used. The rats were raised in the Animal House of the Faculty of Medicine and Biomedical Sciences, University of Yaounde I. They were fed a standard laboratory diet and given tap water ad libitum. They were deprived of food 48 h prior to experimentation, but free access to drinking water was maintained.
2.2. Preparation of the plant extract The fresh bark of V. africana (Yaounde National Herbarium voucher specimen No. HNY/ 1949; P. Nana (collector)) was collected from the campus of the University of Yaounde I in June and oven-dried at 40°C. Dried ground bark (150 g) was extracted exhaustively with methanol for 72 h at room temperature, with occasional stirring. After concentration, the filtered extract solution yielded a reddish residue (30 g) soluble in saline (0.9% NaCl).
2.3. HCl/ethanol-induced ulcers Gastric mucosal ulcers were induced using the method of Hara and Okabe (1985). The test rats were administered the extract of V. africana (500 and 750 mg/kg) while the controls received 1 ml
The effect of pre-treatment with indomethacin on the preventive effect of the extract on HCl/ethanol-induced ulcers was studied following a modification of the method described by Sun et al. (1992). All the rats received indomethacin (20 mg/kg; Merk, Sharp and Dohme, UK) by intraperitoneal route. Later (1 h), the test rats received the extract or sucralfate while the controls received 1 ml of saline orally. This was followed after 1 h by oral administration of 1 ml of the HCl/ethanol solution. The rats were then killed 1 h later using ether and the stomachs observed for ulcers in the glandular portion.
2.5. Pylorus-ligated gastric secretion and ulceration The method of Shay et al. (1945) was used. Following the 48 h fast, the extract and vehicle were administered orally to the test and control rats, respectively, 1 h before the experiment. The pylorus of each rat was tied under light ether anesthesia and the abdominal incisions were closed. The rats were killed 6 h later and the gastric juice produced by each was collected, centrifuged and the volume measured. Ulcers formed in the glandular portion of the stomachs were scored as previously described (Tan et al., 1996).
P.V. Tan et al. / Journal of Ethnopharmacology 73 (2000) 423–428
2.6. Measurement of mucus production Gastric mucus production was measured in rats subjected to pylorus ligation. The mucus covering of each stomach was gently scraped using a glass slide and weighed immediately using a digital precision electronic balance.
2.7. Measurement of gastric acidity The gastric juice obtained from each pylorus ligated animal was centrifuged to obtain a clear solution and the volume measured. A total of 1 ml of centrifuged gastric contents from each rat was assayed for hydrogen ion concentration by pH-metric titration against 0.1 N NaOH using a digital pH meter. Gastric acidity was expressed as mEq/l.
2.8. Measurement of pepsin acti6ity Pepsin activity in the gastric juice collected from each Shay-ligated rat was determined using bovine serum albumin (Sigma) (2.5% w/v in 0.01 M HCl, pH 2) as the substrate. The results were expressed in terms of the amount of liberated L-tyrosine (umol/ml per minute) according to the modified method of Anson and Mirsky described by Rodier and Mallein (1973).
2.9. Statistical analysis Comparisons between treatment means were performed using the Student’s t-test. Values in
425
tables are expressed as arithmetic mean9 S.E.M.
3. Results The bark methanol extract of V. africana (500– 750 mg/kg) dose-dependently offered significant cytoprotection (40–63% inhibition) to the stomach mucosa of the rats against the HCl/ethanol solution compared with the controls. The mean ulcer index score reduced from 5.49 in the controls to 3.28 and 2.00 for the rats receiving 500 and 750 mg/kg of extract, respectively. Sucralfate (100 mg/kg) also significantly inhibited gastric ulceration compared with the controls (Table 1). The inhibitory effect of the extract and sucralfate against HCl/ethanol-induced ulcer were significantly (PB 0.01) suppressed when the rats were pre-treated with indomethacin (Table 2). Tables 3–5 show the results obtained when the animals were subjected to pyloric ligature. Significant decreases of the ulcer index scores were obtained when the extract of V. africana was administered. Ulcer indices were 2.36 and 1.00 for the 500 and 750 mg/kg dose of extract compared with 3.74 for the controls (Table 3). However, the decrease in ulcer index scores in the extracttreated groups were not accompanied by significant decreases in the pH, volume or acidity of gastric juice compared with the controls (Table 4). Gastric mucus production did not differ significantly between extract-treated and control groups. The proteolytic activity of the gastric juice did not change significantly when the extract was administered to the rats followed by pylorus ligation (Table 5).
Table 1 Effect of the methanol extract of V. africana on gastric ulcers induced by HCl/ethanol in rats Treatment
Dose (mg/kg)
Na
Ulcer index (mean 9S.E.M.)
Inhibition (%)
Ulcerated surface (%)
Control Extract Extract Sucralfate
– 500 750 100
6 6 6 8
5.4990.49 3.2890.16* 2.0090.64* 2.9590.30*
– 40.3 63.6 46.3
16.3 7.4 0.6 2.6
a
N, number of rats. * PB0.05, statistically significant relative to control.
P.V. Tan et al. / Journal of Ethnopharmacology 73 (2000) 423–428
426
Table 2 Effect of pre-treatment with indomethacin on the preventive effect of V. africana extract on HCl/ethanol-induced gastric lesions in rats Treatment
Control Extract Extract Sucralfate
Dose (mg/kg)
500 750 100
Controla
Indomethacina,b
Ulcer index (mean9 S.E.M.)
Inhibition (%)
Ulcer index (mean 9 SEM)
Inhibition (%)
−5.019 0.12 3.809 0.16 1.809 0.16* 2.929 0.29*
– 24.1 64.1 41.7
5.43 90.14 5.00 90.13 4.69 9 0.16 5.44 9 0.51
– 7.9 13.6 0.0
a
N= 6 rats per treatment. Rats pre-treated with indomethacin prior to HCl/ethanol treatment. * Statistically significant relative to control, PB0.01.
b
Table 3 Effect of the methanol extract of V. africana on pylorus ligated gastric ulceration in rats Treatment
Dose (mg/kg)
Na
Ulcer index (mean 9S.E.M.)
Inhibition (%)
Ulcerated surface (%)
Control Extract Extract Ranitidine
– 500 750 50
6 6 6 6
3.74 9 0.47 2.36 9 0.61* 1.00 9 0.56* 0.89 9 0.54**
– 36.9 73.3 76.2
14.3 8.4 0.6 0.5
a
N, number of rats. * PB0.05, statistically significant relative to control. ** PB0.01, statistically significant relative to control. Table 4 Effect of V. africana extract on gastric acid secretion in rats Treatment
Dose (mg/kg)
N a Gastric pH (mean 9 S.E.M.)
Volume of gastric juice (ml) (mean 9S.E.M.)
Gastric acidity (mEq/l) (mean9 S.E.M.)
Control Extract Extract Ranitidine
– 500 750 50
6 6 6 6
3.17 90.59 3.38 90.29 3.85 90.85 2.78 90.32
72.8 9 18.4 67.3 9 6.4 60.1 911.5 32.5 9 4.2*
2.989 0.10 2.97 9 0.13 3.149 0.19 3.689 0.09
a
N, number of rats. * PB0.01, statistically significant relative to control.
Table 5 Effect of V. africana extract on gastric mucus production and proteolytic activity in rats Treatment
Dose (mg/kg)
Na
Mucus production (mean 9S.E.M.) Proteolytic activity (umol tyrosine/ml/min)
Control Extract Extract
– 500 750
6 6 6
347.6 955.7 321.2 9 35.2 343.8922.7
a
N, number of rats.
3.17 9 0.59 3.38 90.29 3.85 9 0.85
P.V. Tan et al. / Journal of Ethnopharmacology 73 (2000) 423–428
4. Discussion and conclusions The results of this study confirm the findings of previous studies (Tan et al., 1997a) that showed that the bark aqueous extract of V. africana possesses anti-ulcer properties that are not related to an anti-secretory effect. This suggests that the protection offered by the extract against the HCl/ ethanol solution is not due to a simple neutralization of the HCl in the solution. Although gastric acidity remained high (60 – 67 mEq/l) when the extract was administered to pylorus ligated rats, a significant decrease in ulcer index was obtained. Similar results were obtained when Akhtar and Ahmad (1995) administered the whole plant methanolic extract of Trianthema pentandra to pylorus-ligated rats. In Shay-ligated rats, gastric acid levels between 40 and 65 mEq/l have been associated with severe ulceration of the rat gastric mucosa (Martin et al., 1993; Marhuenda et al., 1993; Tan et al., 1996). Because pepsin and HCl are important for the formation of pylorus ligated ulcers, the protective effect of the extract of V. africana could arguably have been achieved either through increased mucus secretion that would re-enforce gastric mucous defenses, or through the reduction of the proteolytic activity of the pepsin in the gastric juice. This does not appear to be the case, since gastric mucus production and pepsin activity remained unchanged when increasing doses of the extract were administered to the rats. In peptic ulcer therapy, constipation constitutes a frequent side-effect brought about by acid-neutralizing antacids and antisecretory drugs such as Ranitidine. The maintenance of high gastric acidity following oral administration of the extract of V. africana implies that it may not interfere with protein digestion in the stomach, a process that requires an optimum pH range of 1.6 – 3.2. The pH of the gastric juice obtained from the pylorus ligated rats that received the extract were 2.97 9 0.13 and 3.14 90.19 for the 500 and 750 mg/kg doses, respectively. Intraperitoneal administration of indomethacin significantly suppressed the protective effect of the extract against the HCl/ethanol solution. Indomethacin reduces prostaglandin (PG) and bicarbonate secretion, and gastric mucosal blood
427
flow in animals (Whittle, 1977; Flemstrom et al., 1982; Miller, 1982; Selling et al., 1987). The role of PGs in gastric mucosal protection has been discussed in detail by Konturek et al. (1981, 1982); Robert (1981), Miller (1982), Robert et al. (1983). When the cytoprotective effect of an antiulcer agent is significantly reduced by pre-treatment with indomethacin, the cytoprotection is interpreted as being mediated by endogenous PGs. From this view point, therefore, the effects of the extract of V. africana observed in this study may be interpreted similarly. Thus, in the absence of increased mucus production or reduced pepsin activity, the extract may confer cytoprotection through a mechanism involving the physico– chemical re-enforcement of the gastric mucous layer or by effects similar to endogenous PGs.
Acknowledgements This project was supported by the International Foundation for Science (IFS), Stockholm, Sweden, through Grant F/2882-1 (PVT) in collaboration with the Committee on Scientific and Technological Cooperation (COMSTECH) of the Organization of Islamic Conference (OIC), Islamabad, Pakistan.
References Adjanohoun, J.E., Aboubakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., Enow-Orock, E.G., Focho, D., Gbile, Z.O., Kamanyi, A., Kamsu-Kom, J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiele, A.L., Mbome, L.L., Mubiru, N.K., Nancy, W.L., Nkongmeneck, B., Satabie, B., Sofowora, A., Tamze, V., Wirmum, C.K., 1996. Traditional Medicine and Pharmacopoeia: Contribution to Ethnobotanical and Floristic Studies in Cameroon. Organization of African Unity Scientific, Technical and Research Commission. Centre Nationale de Production de Manuels Scolaires, Porto-Novo, pp. 63. Akhtar, A.H., Ahmad, K.U., 1995. Anti-ulcerogenic evaluation of the methanolic extracts of some indigenous medicinal plants of Pakistan in aspirin-ulcerated rats. Journal of Ethnopharmacology 46 (1), 1 – 6. Bouquet, A., Debray, M., 1974. Plantes medicinales de la Cote d’Ivoire. Office de la Recherche Scientifique et Technique d’Outre Mer (ORSTOM) 38 – 39 (165), 11.
428
P.V. Tan et al. / Journal of Ethnopharmacology 73 (2000) 423–428
Flemstrom, G., Garner, A., Nylander, O., Hurst, B.C., Heylings, J.R., 1982. Surface epithelial HCO3 transport by mammalian duodenum in vivo. American Journal of Physiology (Gatrointestinal Liver Physiology) 234, G348– G358. Hara, N., Okabe, S., 1985. Effect of gefernate on acute lesions in rats. Folia Pharmacologica Japonica 85, 443–448. Konturek, S.J., Piastucki, I., Brozozowski, T., Dembiska-kiec, T.R.A., Muda, A., Gryglewski, R., 1981. Role of prostaglandins in the formation of aspirin-induced ulcers. Gastroenterology 80, 4–9. Konturek, S.J., Brozozowski, T., Piastucki, I., Radecki, T., Dembiski-kiec, A., 1982. Role of locally generated prostaglandins in adaptive cytoprotection. Digestive Diseases and Science 27, 967–971. Marhuenda, E., Martin, M.J., Alarcon de la Lastra, C., 1993. Antiulcerogenic activity of Aescine in different experimental models. Phytotherapy Research 7, 13–16. Martin, M.J., Motilva, V., Alarcon de la Lastra, C., 1993. Quercetin and naringenin: effects on ulcer formation and gastric secretion in rats. Phytotherapy Research 7, 150– 153. Miller, T.A., 1982. Protective effect of prostaglandins against gastric mucosal damage: current knowledge and proposed mechanisms. American Journal of Physiology (Gastrointestinal and Liver Physiology 8) 245, G601–G603. Robert, A., 1981. Prostaglandins and the gastroduodenal tract. In: Johnson, L.R. (Ed.), Physiology of the Gastrointestinal Tract. Raven Press, New York, pp. 1407– 1434. Robert, A., Nezamis, J.E., Lancaster, C., Davis, J.P., Field, S.O., Hanchar, A.J., 1983. Mild irritants prevent gastric necrosis through ‘adaptive cytoprotection’ mediated by
.
prostaglandins. American Journal of Physiology (Gastrointestinal and Liver Physiology 8) 245, G113 – G121. Rodier, J., Mallein, R., 1973. In: Maloine, S.A. (Ed.), Manuel de Biochimie Pratque a l’Usage des Laboratoires d’Analyse Medicales, pp. 507 – 510. Selling, J.A., Hogan, D.L., Aly, A., Koss, M.A., Isenberg, J.I., 1987. Indomethacin inhibits duodenal bicarbonate secretion and endogenous prostaglandin E2 output in human subjects. Annals of Internal Medicine 106, 368 – 371. Shay, J.P., Komarov, S.A., Fels, S.S., Meranze, D., Grunstein, M., Simpler, H., 1945. A simple method for the uniform production of gastric ulceration in the rat. Gastroenterology 5, 43 – 61. Sun, Z., Matsumoto, T., Yamada, H., 1992. Anti-ulcer activity and mode of action of the polysaccharide fraction from the leaves of Panax ginseng. Planta Medica 58, 432 – 435. Tan, P.V., Nditafon, G.N., Yewah, M.P., Ayafor, J.F., Dimo, T., 1996. Eremomastax speciosa: effects of leaf aqueous extract on ulcer formation and gastric secretion in rats. Journal of Ethnopharmacology 54, 139 – 142. Tan, P.V., Njimi, C.K., Ayafor, J.F., 1997a. Screening of some African medicinal plants for antiulcerogenic activity. Part I. Phytotherapy Research 11, 45 – 47. Tan, P.V., Lyonga, E.L., Nditafon, G.N., Njimi, C.K., Bopelet, M., 1997b. Gastric cytoprotective antiulcerogenic actions of the aqueous bark extract of Voacanga africana and leaf extract of Eremomastax speciosa in rats. Cameroon Journal of Biological and Biochemical Sciences 7 (1), 69 – 77. Whittle, B.J.R., 1977. Mechanisms underlying gastric mucosal damage induced by indomethacin and bile salts, and the actions of prostaglandins. British Journal of Pharmacology 60, 455 – 460.
Anti-ulcer actions of the bark methanol extract of Voacanga africana in different experimental ulcer models in rats Paul V. Tan a,*, Veronique B. Penlap b, Barthelemy Nyasse c, Joseph D.B. Nguemo b a
Department of Animal Biology and Physiology, Faculty of Science, Uni6ersity of Yaounde I, P.O. Box 812, Yaounde, Cameroon b Department of Biochemistry, Faculty of Science, Uni6ersity of Yaounde I, P.O. Box 812, Yaounde, Cameroon c Department of Organic Chemistry, Faculty of Science, Uni6ersity of Yaounde I, P.O. Box 812, Yaounde, Cameroon Received 26 January 2000; received in revised form 28 May 2000; accepted 6 June 2000
Abstract The antiulcerogenic effects of the bark methanol extract of Voacanga africana were studied using various experimental ulcer models in rats. The effects of the extract on the volume of gastric juice, gastric pH, acid output, mucus production and peptic activity were recorded, as well as the preventive action against lesions caused by HCl/ethanol and indomethacin. Oral administration of the extract (500 – 750 mg/kg) inhibited the formation of gastric lesions induced by HCl/ethanol (40–63% inhibition). The inhibitory effect against HCl/ethanol was significantly (PB 0.01) suppressed by pre-treatment of the rats with indomethacin (30 mg/kg, i.p.). The extract significantly reduced gastric lesion formation in pylorus ligated rats, but this was not associated with an increase in gastric mucus production or with a reduction in acid content, volume of gastric secretion or pepsin activity of the gastric juice. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Voacanga africana; Apocynaceae; Bark methanol extract; Anti-ulcer activity
1. Introduction Voacanga africana Staph. (Apocynaceae) is a tree plant occurring in all of West Africa and as far as the Congo and even Tanzania. In Cote d’Ivoire, leprosy, diarrhoea, generalized oedema, convulsions in children and madness figure among the ethno-medical applications of the plant (Bouquet and Debray, 1974). In Cameroon, the fruit, bark and leaf extracts are used to treat cases of * Corresponding author.
orchitis, ectopic testes and gonorrhoea, respectively (Adjanohoun et al., 1996). Information provided by practitioners of traditional medicine suggest that V. africana also possesses useful antiulcer properties, although this was not cited in the national ethno-botanical survey of Cameroonian medicinal plants conducted by Adjanohoun et al. (1996). In a previous study, the gastric protective effect of the bark aqueous extract of V. africana against HCl/ethanol solution was demonstrated (Tan et al., 1997a). When further tested in the pylorus
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 2 9 2 - 0
424
P.V. Tan et al. / Journal of Ethnopharmacology 73 (2000) 423–428
ligated rat model, the extract prevented gastric mucosal damage, but did not significantly reduce the acidity of gastric juice (Tan et al., 1997b). However, we have observed in previous work (Tan et al., 1996) that gastric acidity within the 50 – 60 mEq/l range causes severe gastric mucosal ulceration. This ulcerative effect of the gastric juice is due to the presence of pepsin and HCl that auto digest the gastric mucosa (Shay et al., 1945). The present paper describes the effects of the bark methanol extract of V. africana in different experimental ulcer models. The possible mode of action of the extract against the pylorus ligated ulcer model was investigated and discussed.
of saline orally. Another group of rats received 100 mg/kg of commercial sucralfate (Usiphar-F; Compeigne, France). The HCl/ethanol solution (150 mM HCl in 60% v/v ethanol) was given 1 h later. The rats were killed after 1 h using ether. The stomachs were removed and cut open along the greater curvature. The length and width of each lesion in the glandular portion was measured and the lesions were scored as described previously (Tan et al., 1996). Percentage ulcerated surface was calculated by expressing the total area (mm2) covered by the lesions as a proportion of the total corpus mucosal surface.
2. Materials and methods
2.4. HCl/ethanol-induced ulcers in rats pre-treated with indomethacin
2.1. Animals Three-month old male albino Wistar rats (160 – 200 g) were used. The rats were raised in the Animal House of the Faculty of Medicine and Biomedical Sciences, University of Yaounde I. They were fed a standard laboratory diet and given tap water ad libitum. They were deprived of food 48 h prior to experimentation, but free access to drinking water was maintained.
2.2. Preparation of the plant extract The fresh bark of V. africana (Yaounde National Herbarium voucher specimen No. HNY/ 1949; P. Nana (collector)) was collected from the campus of the University of Yaounde I in June and oven-dried at 40°C. Dried ground bark (150 g) was extracted exhaustively with methanol for 72 h at room temperature, with occasional stirring. After concentration, the filtered extract solution yielded a reddish residue (30 g) soluble in saline (0.9% NaCl).
2.3. HCl/ethanol-induced ulcers Gastric mucosal ulcers were induced using the method of Hara and Okabe (1985). The test rats were administered the extract of V. africana (500 and 750 mg/kg) while the controls received 1 ml
The effect of pre-treatment with indomethacin on the preventive effect of the extract on HCl/ethanol-induced ulcers was studied following a modification of the method described by Sun et al. (1992). All the rats received indomethacin (20 mg/kg; Merk, Sharp and Dohme, UK) by intraperitoneal route. Later (1 h), the test rats received the extract or sucralfate while the controls received 1 ml of saline orally. This was followed after 1 h by oral administration of 1 ml of the HCl/ethanol solution. The rats were then killed 1 h later using ether and the stomachs observed for ulcers in the glandular portion.
2.5. Pylorus-ligated gastric secretion and ulceration The method of Shay et al. (1945) was used. Following the 48 h fast, the extract and vehicle were administered orally to the test and control rats, respectively, 1 h before the experiment. The pylorus of each rat was tied under light ether anesthesia and the abdominal incisions were closed. The rats were killed 6 h later and the gastric juice produced by each was collected, centrifuged and the volume measured. Ulcers formed in the glandular portion of the stomachs were scored as previously described (Tan et al., 1996).
P.V. Tan et al. / Journal of Ethnopharmacology 73 (2000) 423–428
2.6. Measurement of mucus production Gastric mucus production was measured in rats subjected to pylorus ligation. The mucus covering of each stomach was gently scraped using a glass slide and weighed immediately using a digital precision electronic balance.
2.7. Measurement of gastric acidity The gastric juice obtained from each pylorus ligated animal was centrifuged to obtain a clear solution and the volume measured. A total of 1 ml of centrifuged gastric contents from each rat was assayed for hydrogen ion concentration by pH-metric titration against 0.1 N NaOH using a digital pH meter. Gastric acidity was expressed as mEq/l.
2.8. Measurement of pepsin acti6ity Pepsin activity in the gastric juice collected from each Shay-ligated rat was determined using bovine serum albumin (Sigma) (2.5% w/v in 0.01 M HCl, pH 2) as the substrate. The results were expressed in terms of the amount of liberated L-tyrosine (umol/ml per minute) according to the modified method of Anson and Mirsky described by Rodier and Mallein (1973).
2.9. Statistical analysis Comparisons between treatment means were performed using the Student’s t-test. Values in
425
tables are expressed as arithmetic mean9 S.E.M.
3. Results The bark methanol extract of V. africana (500– 750 mg/kg) dose-dependently offered significant cytoprotection (40–63% inhibition) to the stomach mucosa of the rats against the HCl/ethanol solution compared with the controls. The mean ulcer index score reduced from 5.49 in the controls to 3.28 and 2.00 for the rats receiving 500 and 750 mg/kg of extract, respectively. Sucralfate (100 mg/kg) also significantly inhibited gastric ulceration compared with the controls (Table 1). The inhibitory effect of the extract and sucralfate against HCl/ethanol-induced ulcer were significantly (PB 0.01) suppressed when the rats were pre-treated with indomethacin (Table 2). Tables 3–5 show the results obtained when the animals were subjected to pyloric ligature. Significant decreases of the ulcer index scores were obtained when the extract of V. africana was administered. Ulcer indices were 2.36 and 1.00 for the 500 and 750 mg/kg dose of extract compared with 3.74 for the controls (Table 3). However, the decrease in ulcer index scores in the extracttreated groups were not accompanied by significant decreases in the pH, volume or acidity of gastric juice compared with the controls (Table 4). Gastric mucus production did not differ significantly between extract-treated and control groups. The proteolytic activity of the gastric juice did not change significantly when the extract was administered to the rats followed by pylorus ligation (Table 5).
Table 1 Effect of the methanol extract of V. africana on gastric ulcers induced by HCl/ethanol in rats Treatment
Dose (mg/kg)
Na
Ulcer index (mean 9S.E.M.)
Inhibition (%)
Ulcerated surface (%)
Control Extract Extract Sucralfate
– 500 750 100
6 6 6 8
5.4990.49 3.2890.16* 2.0090.64* 2.9590.30*
– 40.3 63.6 46.3
16.3 7.4 0.6 2.6
a
N, number of rats. * PB0.05, statistically significant relative to control.
P.V. Tan et al. / Journal of Ethnopharmacology 73 (2000) 423–428
426
Table 2 Effect of pre-treatment with indomethacin on the preventive effect of V. africana extract on HCl/ethanol-induced gastric lesions in rats Treatment
Control Extract Extract Sucralfate
Dose (mg/kg)
500 750 100
Controla
Indomethacina,b
Ulcer index (mean9 S.E.M.)
Inhibition (%)
Ulcer index (mean 9 SEM)
Inhibition (%)
−5.019 0.12 3.809 0.16 1.809 0.16* 2.929 0.29*
– 24.1 64.1 41.7
5.43 90.14 5.00 90.13 4.69 9 0.16 5.44 9 0.51
– 7.9 13.6 0.0
a
N= 6 rats per treatment. Rats pre-treated with indomethacin prior to HCl/ethanol treatment. * Statistically significant relative to control, PB0.01.
b
Table 3 Effect of the methanol extract of V. africana on pylorus ligated gastric ulceration in rats Treatment
Dose (mg/kg)
Na
Ulcer index (mean 9S.E.M.)
Inhibition (%)
Ulcerated surface (%)
Control Extract Extract Ranitidine
– 500 750 50
6 6 6 6
3.74 9 0.47 2.36 9 0.61* 1.00 9 0.56* 0.89 9 0.54**
– 36.9 73.3 76.2
14.3 8.4 0.6 0.5
a
N, number of rats. * PB0.05, statistically significant relative to control. ** PB0.01, statistically significant relative to control. Table 4 Effect of V. africana extract on gastric acid secretion in rats Treatment
Dose (mg/kg)
N a Gastric pH (mean 9 S.E.M.)
Volume of gastric juice (ml) (mean 9S.E.M.)
Gastric acidity (mEq/l) (mean9 S.E.M.)
Control Extract Extract Ranitidine
– 500 750 50
6 6 6 6
3.17 90.59 3.38 90.29 3.85 90.85 2.78 90.32
72.8 9 18.4 67.3 9 6.4 60.1 911.5 32.5 9 4.2*
2.989 0.10 2.97 9 0.13 3.149 0.19 3.689 0.09
a
N, number of rats. * PB0.01, statistically significant relative to control.
Table 5 Effect of V. africana extract on gastric mucus production and proteolytic activity in rats Treatment
Dose (mg/kg)
Na
Mucus production (mean 9S.E.M.) Proteolytic activity (umol tyrosine/ml/min)
Control Extract Extract
– 500 750
6 6 6
347.6 955.7 321.2 9 35.2 343.8922.7
a
N, number of rats.
3.17 9 0.59 3.38 90.29 3.85 9 0.85
P.V. Tan et al. / Journal of Ethnopharmacology 73 (2000) 423–428
4. Discussion and conclusions The results of this study confirm the findings of previous studies (Tan et al., 1997a) that showed that the bark aqueous extract of V. africana possesses anti-ulcer properties that are not related to an anti-secretory effect. This suggests that the protection offered by the extract against the HCl/ ethanol solution is not due to a simple neutralization of the HCl in the solution. Although gastric acidity remained high (60 – 67 mEq/l) when the extract was administered to pylorus ligated rats, a significant decrease in ulcer index was obtained. Similar results were obtained when Akhtar and Ahmad (1995) administered the whole plant methanolic extract of Trianthema pentandra to pylorus-ligated rats. In Shay-ligated rats, gastric acid levels between 40 and 65 mEq/l have been associated with severe ulceration of the rat gastric mucosa (Martin et al., 1993; Marhuenda et al., 1993; Tan et al., 1996). Because pepsin and HCl are important for the formation of pylorus ligated ulcers, the protective effect of the extract of V. africana could arguably have been achieved either through increased mucus secretion that would re-enforce gastric mucous defenses, or through the reduction of the proteolytic activity of the pepsin in the gastric juice. This does not appear to be the case, since gastric mucus production and pepsin activity remained unchanged when increasing doses of the extract were administered to the rats. In peptic ulcer therapy, constipation constitutes a frequent side-effect brought about by acid-neutralizing antacids and antisecretory drugs such as Ranitidine. The maintenance of high gastric acidity following oral administration of the extract of V. africana implies that it may not interfere with protein digestion in the stomach, a process that requires an optimum pH range of 1.6 – 3.2. The pH of the gastric juice obtained from the pylorus ligated rats that received the extract were 2.97 9 0.13 and 3.14 90.19 for the 500 and 750 mg/kg doses, respectively. Intraperitoneal administration of indomethacin significantly suppressed the protective effect of the extract against the HCl/ethanol solution. Indomethacin reduces prostaglandin (PG) and bicarbonate secretion, and gastric mucosal blood
427
flow in animals (Whittle, 1977; Flemstrom et al., 1982; Miller, 1982; Selling et al., 1987). The role of PGs in gastric mucosal protection has been discussed in detail by Konturek et al. (1981, 1982); Robert (1981), Miller (1982), Robert et al. (1983). When the cytoprotective effect of an antiulcer agent is significantly reduced by pre-treatment with indomethacin, the cytoprotection is interpreted as being mediated by endogenous PGs. From this view point, therefore, the effects of the extract of V. africana observed in this study may be interpreted similarly. Thus, in the absence of increased mucus production or reduced pepsin activity, the extract may confer cytoprotection through a mechanism involving the physico– chemical re-enforcement of the gastric mucous layer or by effects similar to endogenous PGs.
Acknowledgements This project was supported by the International Foundation for Science (IFS), Stockholm, Sweden, through Grant F/2882-1 (PVT) in collaboration with the Committee on Scientific and Technological Cooperation (COMSTECH) of the Organization of Islamic Conference (OIC), Islamabad, Pakistan.
References Adjanohoun, J.E., Aboubakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., Enow-Orock, E.G., Focho, D., Gbile, Z.O., Kamanyi, A., Kamsu-Kom, J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiele, A.L., Mbome, L.L., Mubiru, N.K., Nancy, W.L., Nkongmeneck, B., Satabie, B., Sofowora, A., Tamze, V., Wirmum, C.K., 1996. Traditional Medicine and Pharmacopoeia: Contribution to Ethnobotanical and Floristic Studies in Cameroon. Organization of African Unity Scientific, Technical and Research Commission. Centre Nationale de Production de Manuels Scolaires, Porto-Novo, pp. 63. Akhtar, A.H., Ahmad, K.U., 1995. Anti-ulcerogenic evaluation of the methanolic extracts of some indigenous medicinal plants of Pakistan in aspirin-ulcerated rats. Journal of Ethnopharmacology 46 (1), 1 – 6. Bouquet, A., Debray, M., 1974. Plantes medicinales de la Cote d’Ivoire. Office de la Recherche Scientifique et Technique d’Outre Mer (ORSTOM) 38 – 39 (165), 11.
428
P.V. Tan et al. / Journal of Ethnopharmacology 73 (2000) 423–428
Flemstrom, G., Garner, A., Nylander, O., Hurst, B.C., Heylings, J.R., 1982. Surface epithelial HCO3 transport by mammalian duodenum in vivo. American Journal of Physiology (Gatrointestinal Liver Physiology) 234, G348– G358. Hara, N., Okabe, S., 1985. Effect of gefernate on acute lesions in rats. Folia Pharmacologica Japonica 85, 443–448. Konturek, S.J., Piastucki, I., Brozozowski, T., Dembiska-kiec, T.R.A., Muda, A., Gryglewski, R., 1981. Role of prostaglandins in the formation of aspirin-induced ulcers. Gastroenterology 80, 4–9. Konturek, S.J., Brozozowski, T., Piastucki, I., Radecki, T., Dembiski-kiec, A., 1982. Role of locally generated prostaglandins in adaptive cytoprotection. Digestive Diseases and Science 27, 967–971. Marhuenda, E., Martin, M.J., Alarcon de la Lastra, C., 1993. Antiulcerogenic activity of Aescine in different experimental models. Phytotherapy Research 7, 13–16. Martin, M.J., Motilva, V., Alarcon de la Lastra, C., 1993. Quercetin and naringenin: effects on ulcer formation and gastric secretion in rats. Phytotherapy Research 7, 150– 153. Miller, T.A., 1982. Protective effect of prostaglandins against gastric mucosal damage: current knowledge and proposed mechanisms. American Journal of Physiology (Gastrointestinal and Liver Physiology 8) 245, G601–G603. Robert, A., 1981. Prostaglandins and the gastroduodenal tract. In: Johnson, L.R. (Ed.), Physiology of the Gastrointestinal Tract. Raven Press, New York, pp. 1407– 1434. Robert, A., Nezamis, J.E., Lancaster, C., Davis, J.P., Field, S.O., Hanchar, A.J., 1983. Mild irritants prevent gastric necrosis through ‘adaptive cytoprotection’ mediated by
.
prostaglandins. American Journal of Physiology (Gastrointestinal and Liver Physiology 8) 245, G113 – G121. Rodier, J., Mallein, R., 1973. In: Maloine, S.A. (Ed.), Manuel de Biochimie Pratque a l’Usage des Laboratoires d’Analyse Medicales, pp. 507 – 510. Selling, J.A., Hogan, D.L., Aly, A., Koss, M.A., Isenberg, J.I., 1987. Indomethacin inhibits duodenal bicarbonate secretion and endogenous prostaglandin E2 output in human subjects. Annals of Internal Medicine 106, 368 – 371. Shay, J.P., Komarov, S.A., Fels, S.S., Meranze, D., Grunstein, M., Simpler, H., 1945. A simple method for the uniform production of gastric ulceration in the rat. Gastroenterology 5, 43 – 61. Sun, Z., Matsumoto, T., Yamada, H., 1992. Anti-ulcer activity and mode of action of the polysaccharide fraction from the leaves of Panax ginseng. Planta Medica 58, 432 – 435. Tan, P.V., Nditafon, G.N., Yewah, M.P., Ayafor, J.F., Dimo, T., 1996. Eremomastax speciosa: effects of leaf aqueous extract on ulcer formation and gastric secretion in rats. Journal of Ethnopharmacology 54, 139 – 142. Tan, P.V., Njimi, C.K., Ayafor, J.F., 1997a. Screening of some African medicinal plants for antiulcerogenic activity. Part I. Phytotherapy Research 11, 45 – 47. Tan, P.V., Lyonga, E.L., Nditafon, G.N., Njimi, C.K., Bopelet, M., 1997b. Gastric cytoprotective antiulcerogenic actions of the aqueous bark extract of Voacanga africana and leaf extract of Eremomastax speciosa in rats. Cameroon Journal of Biological and Biochemical Sciences 7 (1), 69 – 77. Whittle, B.J.R., 1977. Mechanisms underlying gastric mucosal damage induced by indomethacin and bile salts, and the actions of prostaglandins. British Journal of Pharmacology 60, 455 – 460.