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Anti-ulcer compound from Voacanga africana with possible histamine H2 receptor blocking activity
Phytomedicine , Vol. 7(6), pp. 509-515
© Urban & Fischer Verlag 2000 http:// www.urbanfischer.deliournals/phytomed
Anti-ulcer compound from Voacanga africana with possible histamine H2 receptor blocking activity P. V. Tan 1 and B. Nyasse! IDepartment of Animal Biology and Physiology, 2Department of Organic Chemi stry, Faculty of Science, Univer sity of Yaounde I, Cameroon
Summary Voacanga africana is used in Cameroonian ethnomedicine for the treatment of peptic ulcers. We have tested the cytoprotective, anti-secretory and ulcer healing actions of an alkaloid (TN) obtained from the fruit extract. Oral administration of TN (50-100 mg/kg) dose-dependently prevented ulcer formation by HCl/ethanol (36-75 % ), absolute ethanol (43-75 %), HCI-ethanol/indomethacin (58-84 %), Pylorus ligation (31-100 %) , cold restraint stress (68-100 %) and histamine (49-100 %). The inhibitory effect at SO and 100 mg/kg against HCl/ethanol was not suppressed by pre-treatment with indomethacin (20 mg/kg, i.p.), TN reduced Shay-ligated gastric acid secretion from 77 mEq/1 in the controls to 46 and 25 mEq/1 for the 50 and 100 mg/kg doses. Augmented histamine-induced gastric acid secretion was reduced from 84 mEq/1 in the controls to 45 and 21 mEq/1 for the two doses of TN, with total inhibition of gastric and duodenal ulcers by the 50 mg/kg dose. Healing rate of chronic acetic acid-induced ulcers was 62 and 83 %, respectively, for the dose of 50 and 100 mg/kg of TN compared with the controls. TN has gastric anti-secretory effects similar to histamine receptor blockers. Its cytoprotective and ulcer healing properties are related to its ability to strengthen gastric mucosal defenses through enhanced gastric mucus production. Key words: Voacanga africana, anti-ulcer activity, gastric secretion, str ess ulcer.
Introduction The discovery in the mid nineteen seventies of anti-secretor y agents capable of low ering the abnormally high acid secretion rates in pat ients marked a turning point in peptic ulcer disease management and treatment (Hall, 1997). Gastric acid hyper secretion th at causes peptic ulcer th rou gh gastric mucosal autodigestion can be of str ess or genetic origin as well as th e result of the interaction of a genetic component with en vironmental factors (Grossma n, 1981 ). The more recent discovery of the role of H elicobacter pylori in the etiology of peptic ulcer disease has led to a radical cha nge in the classical method of treatment and/or management of gastro-du odenal ulcers (Dixon et al., 1996). Thus, the triple th erap y regimen currently recommended for gastric hyper secretors includes a mixture of antacids, antisecretory agents and antibiotics. In Cameroon, antisecretory agents constitute a major
detriment to effective compliance to the triple therapy regimen due to their prohibitive costs. The resultant has been a dramatic 50 % drop in the rate of ho spital con sultations. This situatio n highlights the need to inten sify the search for ant isecreto ry materials from local medicinal sources. We have demonstrat ed the anti-ulcer properties of the bark aqueous and methanol extracts of Voacanga africana in rats (Tan et al., 199 7a , b, c) as well as an antimicrobial action against sta phylococcus aureus . We ha ve also shown a cytoprot ective action of the fru it aqueous extract of the plant. The latter results have prompted us to pursue fur th er work on the fruit extract since it is a plant part, unlik e the bark, whose exploitation would pose less threat to the survival of the species. V. africana ha s been listed in Cameroon as a useful but endangered plant species. This tree plant is 0944-7113/00/07/06-509 $ 15.00/0
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P. V. Tan and B. Nya sse ride, Tween-Zf and Dimeth yl sulphoxide (DMSO) were from Sigma Chemical Co., St Louis, USA.
widely occurring in all of West Africa, as far as Congo and even Tanzania (Bouquet and Debra y, 1974). Although a recent national ethno botanical survey of Cameroonian plants used in ethno medicine cited V. africana as a useful medicinal plant for the treatment of ectopic testis, orchiti s and gonorrhea (Adjanohoun et al., 1996 ), anti-ulcer activity was not listed as one of its medicina l properties. The medicinal uses cited elsewhere include toothache, leprosy, diarrhoea, generalized oedema and convulsions in children (Dalziel, 193 7; Bouquet and Debray, 1974). Previous phyto chemical work cited by Bouquet and Debray (1974) shows that the grain extract contains 1 alkaloid while the bark and leaf extracts contain 23 and 13 alkaloids, respectively. The total alkaloids are known to possess cardiotonic and hypotensive properties (Quevauvillier et al., 1955). The gastric cytoprotective anti-ulcerogenic and ulcer healing actions of TN, a compound obtained from the fruit extract, are therefore novel findings that we now report. The possible modes of action of the compound are discussed in relation to the pathogenic mechanisms of action of the vario us necrotizing agents used.
Animals
Ma le Wistar rats (160-200g) raised in the animal house of the Faculty of Medicine and Biomedical Sciences, University of Yaounde I, were used. The y were fed a standard laboratory diet (S.P.c. Ltd, Bafoussam, Cameroon) and given fresh water ad libitum. Before the experiments, they were starved for 48 h in wire mesh bottom cages to prevent coprophagy but allowed free access to water. Preparation of the anti-ulcer compound
The mature fruits of V. ajricana were harvested in Yaounde in August/September 1998 and sun dried . Voucher specimen No. HNC/1949 (P. Nana: Collector), has been deposited at the National Herbarium, Yaounde. A 1:1 mixture of methylene chloride and methanol (5 litres) was used to extract 5 kg of dried ground powder. The resulting solid (300 g) was fractionated by bioassay-guided procedure to obtain 5 g of a pure active component (TN) . TN was identified by the Meyer and FeCl3 tests as an alka loid NOT containing phenolic groups . The compound has a melting point of 192 -195°C and an [a]D 305 0 [c, 0.1; MeOH]. 50 mg of TN were dissolved in 3 to 5 drops of DMSO and the solution made up to 100 ml using a 0.25 % solution of Tween-20. A 1 % solution of DMSO in 0.25 % Tween-20 was prepared and used as the vehicle control.
Material and methods Material
Sucralfate (Usiphar-F (Compeigne-France), Cimetidine (Smith-Kline & French, 92090 Paris La Defense, France), Indomethacin (Merk Sharp & Dohme, Great Britain) and Ranitidine (Glaxo Wellcome) were purchased from local pharmacies. Histamine dihydrochlo-
Table 1. Effect of TN on gastr ic ulcers induced by HCUethan ol in rats . Treatment Control TN TN Sucralfate
Dose (mg/kg) 50 100 100
N
6 6 6 8
Ulcer index (mean:!:SEM)
Inhibition
5.49 :!: 0.49 3.54 :!: 0.54 * 1.35 :!: 0.64 ** 2.97:!: 0.36 *
(%)
Mucus production (mg)
36 75 50
96.9:!: 102.2:!: 116.5 :!: 48 .0 :!:
7.9 35.5 * 19.9 * 8.1
Statistical significance relative to control, * p < 0.05 ; <. * p < 0.01; N, number of rat s.
Table 2. Effect of TN on gastric ulcers induced by absolute ethan ol in rats . Treatment
Dose (mg/kg)
Control
TN TN Sucralfate
50 100 200
N 6 6 6 8
Ulcer index (mean:!:SEM)
(%)
Mucu s production (mg)
4.88 2.83 1.20 4.49
43 75 8
124 .2:!: 17.6 114.6:!: 16.1 131.8:!: 15.2 " 55.6 :!: 10.7
:!: :!: :!: :!:
0.12 0.89 * 0.81 ** 0.46
Statistical significance relat ive to control, P < 0.05 ; ""p < 0.01 N, number of rats .
Inhibition
Anti-ulcer compound from Yoacanga africana Ulcer preventive tests:
• HCLIethanol-induced gastric mucosal membrane lesions: Gastric muco sal lesions were induced by the method of Hara and Okabe (1985). The test rats were administered the test drug s while the controls received, orall y, 1 ml of the HClJethanol solution (150 mM HCI in 60 % v/v ethanol). The rat s were killed 1 h later using ether and the stomachs remo ved and observed for lesions in the glandular portion. The area covered by each lesion was measured and the lesions were scored as described previously (Tan et al., 199 7a). • Absolute ethanol-induced gastric membrane lesions: Absolute ethanol-induced lesions were provoked using the method described above for the HClIEthanol-induced lesions. The rats received the test drugs or vehicle followed 1 h later by 1 ml of absolute ethanol by oral route. They were also killed using ether and the lesions formed were observed and scored. • HCVEthanol-induced lesions in rats pre-treated with indomethacin: The effect of pre-treatment with indomethacin on the preventive effect of the test drugs on HCllEthanol-induced gastric lesions was studied following a modification of the method described by Sun et al. (1991). All the rats received indomethacin (20 mg/kg) by intraperitoneal route. 1 h later, the test rats received TN or Sucralfate while the controls received 1 ml of vehicle by oral route. Thi s was followed another hour later by oral admini stration of 1 ml of the HClIethanol solution. The rats were then killed 1 h later using ether and the stomachs observed for lesions. • Pylorus -ligated gastric secretion and ulceration: Following a 48 h fast, the test drugs and vehicle were administered orally to the test and control rats, respec-
511
tively, 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 aI., 1996). • Cold restraint stress-induced gastric lesions: Stress-induced gastric lesions were provoked by placing the rats in stress cages and immersing in cold water (3-5 "C) to the level of the xiphoid process for 90 min. The animals received the test products and vehicle 30 min. prior to immersion in cold water. They were killed by a blow to the head and the stomachs opened and observed for lesions in the glandular portion. • Augmented histamine-induced gastric secretion and ulceration: A modified augmented histamine test was used to study the possible mechanism of anti-secretory activity of TN. Control and test animals received vehicle and TN, respectively, 30 min. before pylorus ligation. Repeated hourly injections of histamine dihydrochloride (1 mg/kg, s.c., x 4) were used in combination with the pylorus-ligated ulcer technique to provoke gastric hypersecretion and ulceration. The gastric juice accumulated in the stomach of each animal was collected 1 h after the last histamine injection and assayed for acid content. • Measurement of acid content of gastric juice: Samples of gastric contents (1 rnl) were analyzed for hydrogen ion concentration by pH-metric titration with 0,1 N NaOH solution using a digital pH meter. The acid content was expressed as mliq/l.
Table 3. Effect of TN on gastric ulcers induced by HClIethanol in rats pre-treated with indomethacin. Treatment Control TN TN Sucralfate
Dose (mg/kg) 50 100 100
N
Ulcer index (mean±SEM)
6 6 6 8
5.13 ± 0.14 2.18 ± 0.30 * 0.80 ± 0.50 *,. 6.77 ± 2.40
Inhib ition (%)
Mucus production (mg) 96.0 ± 0.7 118.2 ± 11.8 * 177.4 ± 40.3 " 47.8 ± 9.3
58 84
o
Statistical significance relative to control, *p < 0.05; * "p < 0.01; N , number of rats.
Table 4. Effect of TN on gastric secretion and ulceration in the rats. Treatment
Dose (mg/kg)
Control
TN TN Cimetidine
50 100 50
N
Gastric acidity (rnliq/l ) (mean±SEM)
ulcer index (mean±SEM)
6 6 6 12
77.20 :!: 5.80 47.45 ± 6.72 * 24.91 :!: 4.83"" 72.10:!:11.53
3.15 ± 2.18 ± 0.00 ± 1.10 ±
Statistical significance relative to control,
"p < 0.05; ""p < 0.01; N, number of rats.
0.69 0.68 * 0.00 " '" 0.29 *"
Inhibition (% ) 31 100 68
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P. V. Tan and B. Nyasse
Ulcer healing test:
• Acetic acid-induced chronic gastric ulceration: The method of Takagi et al. described by Yamamoto et al. (1992) was used. The rat stomachs were exteriorized under light ether anaesthesia and the surface of the anterior wall of the glandular stomach was brought into contact with 1 ml of glacial acetic acid using a cylinder (10 mm in diameter) for 30 sec. The test animal s and controls received the test drugs and vehicle, respectively, by oral route for 10 days starting from the fourth day after the acetic acid treatm ent. All the anim als received food and wat er normally during the treatment period. They were depriv ed of food overnight after the last dose of the treatment and sacrificed the next day using ether. The stomachs were removed and examin ed for ulcers. The length and width of each ulcer was measured and the product (mm-) was taken as the ulcer index. Healing rates were then calculated by comparing test animal ulcer indices with the contro ls. • Measurement of mucus production: Gastric mucus production was measured in the rats subjected to
HCVethanol-, absolute ethanol-, indomethacinIHCIethanol- and acetic acid-induced lesions. The gastric muco sa of each rat was gently scraped using a glass slide and the mucus obtained was weighed using a precision electronic balanc e. The same experimenter performed this operation each time. • Statistical analysis: Values in tables are given as arithmetic mean s ± standard error of the mean (S.E.M .) The significance of differences between mean s was calculated using the student's z-test.
Results Oral administration of TN at the dose of 50 and 100 mg/kg dose-dependently prevented the formation of gastric lesions induced by the HCl/ethanol solution. Ulcer index scores reduced from 5.49 in the controls to 1.35 when 100 mg/kg of TN was used. Sucralfate (100 mg/kg) also inhibited th e formation of gastric mucos al lesions, but it was less effective (49 % inhibition ) than
Table 5. Effects of TN on histamin e-indu ced gastro-duodenal ulcers and gastric secretion in rats. Dose (mg/kg)
Treatm ent Contro l TN TN Ranitidine
25 50 100
N
Gastric acidity (mEqll)
Gastric ulcer (mean±SEM)
Du odenal ulcer (mean±SEM)
Inh ibiti on (%) .
6 6 6 6
84 .37 45.68 20 .83 5.83
1.93 ± 0.22 0.86 ± 0.0 9" 0.00 ± 0.00 " " 0.00 ± 0.00 **
1.40 0.79 0.00 0.00
49$ 100 100
± 14.79 ± 04 .68* ± 02.38** ± 0.36 " *
± 0.4 3 ± 0.4 1* ± 0.00*" ± 0.0 0* "
Stat istical significance relative to contro l, *p < 0.05; "" p < 0.01 ; N , number of rats. $ Value is average of gastr ic and duodenal inhibition.
Table 6. Effect of TN on cold restraint stress-induced gastric lesion s in rats. Treatment
N
Dose (rug/k g)
Co ntro l TN TN Ran itidine
6
6 6
25 50 50
12
ulcer index (mean ± SEM )
Inhibition (%)
3.81 1.24 0.00 0.00
68 100 100
± ±
± ±
0.48 0.22 * 0.00 ** 0.00**
Statistical significance relative to cont rol , "p < 0.05; * *p < 0.01; N, number of rats.
Table 7. Effect of TN on th e healing rate of gastric ulcers induced by acetic acid in rat s. Treatm ent Cont rol TN TN Sucralfate
Do se (mglkg) 50 100 100
N
Ulcer index (mea n±SEM)
Heal ing rate (%)
Mucus production (mg)
6 6 6 6
346 .40 ± 131. 80 ± 57.41 ± 107.04 ±
62 83 69
36.9 1± 3.02 83 .73 ± 7.96 " 104.65 ± 1.96 ** 42.25 ± 2.35
32.93 29 .60 * 19.02 " ** 36. 10*
Statistical significance relative to control, *p < 0.05; " "p < 0.01 ; *"" p < 0.001; N, number of rats.
Anti-ulcer compound from Voacanga africana
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TN at the same dose. TN promoted higher levels of healing of acetic acid-induced chronic gastric ulcers (62 mucus production than Sucralfate compared with the to 83 % ) following dail y treatment with TN (50 and 100 mg/kg, respectively) for 10 days. A healing rate of controls (Table 1) 69 % was recorded for Sucralfate (100 mg/kg). Unlike The inhibitory effect of TN at 50 and 100 mg/kg against HCl/ethanol was not suppressed by pre-treat- Sucralfate, TN promoted significantly higher levels of ment with indomethacin. This was not the case with mucus production (83 to 104 mg) during the treatment Sucralfate whose preventive effect (49 %) against period compared with the controls (37 mg ). HCI/ethanol disappeared completely following pretreatment with indomethacin (Table 3). Mucus production increased in a dose-dependent manner when inDiscussion creasing doses of TN were given to the rats. Sucralfate promoted lower mucus production compared with the The anti-ulcer methods were selected in order to test TN for gastric cytoprotective and anti-secretory activicontrols following indomethacin pre-treatment. Table 2 shows the results obtained when absolute ty, as well as protection against ulcers of cold stress oriethanol was used as the necrotizing agent. Gastric le- gin. The HCI/ethanol and absolute ethanol methods sions observed were macro morphologically similar to were used to screen the compound for gastric cytoprothose obtained using the HClIethanol solution. Oral tection against the two mucosal irritant substances, administration of TN at doses of 50 and 100 mg/kg and pre-treatment with indomethacin was used to test dose-dependently prevented the formation of gastric le- if cytoprotection was endogenous prostaglandinsions induced by absolute ethanol (% inhibition = 42 linked. Oral administration of TN to the rats preventand 75, respectively). Sucralfate at the dose of 200 ed the formation of gastric lesions induced by mg/kg was not effective aga inst absolute ethanol (% HCI/ethanol, absolute ethanol, indomethacin/HClinhibition =7.9). Neither TN nor Sucralfate significant- ethanol, cold restraint stress, pylorus ligation, and sigly improved mucus production in absolute ethanol- nificantly enhanced the healing of chronic ulcers intreated rats compared with the controls (Table 2). duced using acetic acid. The gastric cytoprotective efTable 4 shows the results obtained after subjecting fect of TN against absolute ethanol suggests that its the rats to pyloric ligature. Lesions produced were ei- protective action in HCI/ethanol-treated model was rether pointed or raised inflammations unlike the lated to an effect other than the simple neutralization stripped lesions caused by absolute ethanol or of the acid in the necrotizing mixture. A proposed HCI/ethanol. TN (50 mg/kg) produced significantly model for the pathogenesis of ethanol-induced gastric lower ulcer index scores (2.18) compared with the con- mucosal damage in rats involves superficial aggressive trols (3.15), and completely inhibited lesion formation cellular necrosis as well as the release of tissue-derived at the dose of 100 mg/kg. Cimetidine (50 mg/kg) also mediators such as histamine and Leucotriene C4 • These reduced ulcer index scores and % inhibition (67.9) was mediators act on gastric microvasculature, triggering a higher than for TN at the same dose. The gastric juice series of events that lead to mucosal and possibly subobtained 6 h after pylorus ligation was highly acidic mucosal tissue damage (Oates and Hakkinen, 1988). (77 mliq/l) in the control rats although the volumes of The cytoprotective action of TN may, therefore, ingastric contents were similar for all the treatments. TN volve a blockade of the generation of these mediators (50 and 100 mg/kg) dose-dependently reduced gastric or their action on microvasculature. Such a blockade of acidity (46 and 25 mEq/l, respectively) unlike Cimeti- histamine would also be responsible for the antisecredine. The augmented histamine-induced gastric acid se- tory activity of TN observed in the pylorus ligated ulcretion was reduced from 84 mEg/1 in the control rats cer model. to 45 and 21 mEg/1 for the rats that received 25 and 50 Treatment with indomethacin causes gastric mucosal mg/kg of TN, respectively. Neither gastric nor duode- injury through a reduction of endogenous prostaglannal ulcers were observed in animals that received TN din (PG) synthesis, inhibition of gastroduodenal bicar(50 mg/kg) or Ranitidine (100 mg/kg) (Table 5 ). bonate secretion, disruption of the mucosal barrier and The effects of subjecting the rats to a combination of reduction of mucosal blood flow (Whittle, 1977; Sellrestraint and cold stress are shown in Table 6. The con- ing et aI., 1987; Flemstrom et aI., 1982; Miller et aI., trol rats had hemorrhagic lesions in the glandular 1982). However, pre-treatment with indomethacin pristomachs 90 min. after cold immersion. TN (25-50 or to HCI/ethanol did not affect the previously obmg/kg) dose-dependently prevented lesion formation, served effect of TN. Similar results have been interpretwith complete inhibition of lesion formation at the ed by Yamamoto et al. (1992) and Sun et al. (1992) to dose of 50 mg/kg. Gastric and duodenal lesions were mean that the cytoprotective action of the compound also absent in rats given 100 mg/kg of Ranitidine. involves a direct mucosal protection similar to that of Table 7 shows a dose-d ependent enhancement of the PGs and not related to endogenous PGs. PGs are
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P. V. Tan and B. Nyasse
known to prevent experimentally-induced ulcers and to protect the gastro-duodenal mucosa against various ulcerogens. Gastric mucosal protection by TN may involve an enhancement of mucus production as was observed in HClIethanol, HClIethanol/indomethacin and acetic acid ulcer models. Sucralfate, a mucosal strengthener known to stimulate mucus, bicarbonate and PG secretion (Rang and Dale, 1991) did not significantly enhance mucus production compared with TN. The ability of TN to inhibit gastric acid secretion was tested using the pylorus ligation method and the possibility that the observed reduction in gastric acidity could be due to a blockade of histamine receptors was tested using the augmented histamine test. Further more, since ulceration due to cold stress is also histamine linked, activity against stress ulcer was tested when the augmented histamine test gave positive results. Shay's pylorus-ligated ulcer technique also demonstrated the anti-ulcer effect of TN, with complete inhibition of ulcer formation at the dose of 100 mg/kg accompanied by a significant reduction in gastric acidity to 25 mEq/l. Similar levels of gastric acidity have previously been observed not to cause gastric rnucosallesions in pylorus ligated rats (Tan et al., 1996). Pepsin and HCI are important for the formation of pylorus-ligated ulcers (Shay et al., 1945). Thus, the significant reduction in gastric acidity suggests that the cytoprotective action of TN may be exercised through an anti-secretory effect similar to that of Hz blockers of histamine, in addition to a possible direct mucosal protection against mucosal autodigestion by pepsin. TN reduced, significantly, the volume and acid concentration of histamine-induced gastric hyperacidity, preventing the formation of both gastric and duodenal ulcers compared with the controls. Such anti-histaminic effects may also explain the total inhibitory action of TN against cold restraint stress-induced ulcer (Senay and Levine, 1967) since mast cells are known to degranulate and release histamine in response to stress (Guth and Hall, 1966). In addition, TN may also act through scavenger activity, on singlet oxygen, the highly reactive mutagenic oxygen species implicated (through lipid peroxydation) in the mechanism of water immersion stress-induced gastric ulcer (Yoshikawa and Miyagawa, cited by Yamamoto et al., 1992). This possibility was, however, not tested in the present work. The ulcer-healing test also showed a dose-dependent enhancement of the healing of chronic ulcers induced using glacial acetic acid. This was associated with improved mucus production, which can protect the rat stomach mucosa against its own peptic secretions and enhance the healing process. Increased gastric mucosal blood flow, and increased oxygen and nutrient supply to the ulcerated area, which have been cited (Svanes et al., 1991; Hirose et al., 1991) as contributing mecha-
nisms to the healing of acetic acid-induced ulcer, may have been implicated in the healing effect of TN but these were not measured. In conclusion, our results suggest that TN taken orally in experimental ulcer models, has cytoprotective, anti-secretory and healing properties which may explain the clinical effect of the fruit extract of V. africana when employed in ethnomedicine for the management of complaints symptomatic of peptic ulcer disease. These effects are likely due to the ability of TN to strengthen gastric mucosal defenses through enhanced mucus production as well as an anti-secretory effect similar to that of Hz receptor antagonists of histamine. TN appears to be a useful compound for the development of new anti-ulcer drugs and its toxicity profile and detailed mechanism of action await further studies. 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, and Grant F/2626-2 (BN).
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tion of the cat gastric mucosa. Gastroenterology 100: 1249 -1258, 1991. Tan, P.Y., Nditafon, G.N., Yewah, M.P., Ayafor, J.F. and Dimo, T: Eremomastax speciosa: effects of leaf aqueous extract on ulcer formation and gastric secretion in rats. ]. Ethnopharm. 54: 139-142, 1996. Tan, P.Y., Njimi, G.K., and Ayafor, J.F.: Screening of some African medicinal plants for anti ulcerogenic activity. Part I. Phytother. Res. 11: 45-47, 1997a. Tan, P.V., Lyonga, E.L., Nditafon, G.N., Njimi, cx., Bope1et, M.: Gastric cytoprotective antiulcerogenic actions of the aqueous bark extract of Voacanga africana and leaf extract of Eremomastax speciosa in rats. Cam ]. BioI. Biochem Sc. 7 (1): 69-77, 1997b. Tan, P.Y., Penlap, Y.B and Nguemo, J.: Possible mode of action of the methanolic extract of Voacanga africana in pylorus ligated ulcer model. Proc. 5th Annual Conf. of Cam. Biosc. Soc.: Biosciences and Food Security. 17-19 December, Yaounde, Cameroon. pp.155-158, 1997c. Whittle, B.J.R.: Mechanisms underlying gastric mucosal damage induced by indomethacin and bile salts, and the actions of prostaglandins. Br.]. Pharmacal. 60: 455-460, 1977. Yamamoto, K., Kakegawa, H., Matsumoto, H., Sudo, T and Satoh, T.: Gastric cytoprotective anti ulcerogenic actions of hydroxychalcones in rats. Planta Medica 58: 389-393, 1992.
Address P. V. Tan, Animal Physiology laboratory, Department of Animal Biology and Physiology, Faculty of Science, P.O. Box 812, University of Yaounde I, Cameroon. e-mail: [email protected]
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Anti-ulcer compound from Voacanga africana with possible histamine H2 receptor blocking activity P. V. Tan 1 and B. Nyasse! IDepartment of Animal Biology and Physiology, 2Department of Organic Chemi stry, Faculty of Science, Univer sity of Yaounde I, Cameroon
Summary Voacanga africana is used in Cameroonian ethnomedicine for the treatment of peptic ulcers. We have tested the cytoprotective, anti-secretory and ulcer healing actions of an alkaloid (TN) obtained from the fruit extract. Oral administration of TN (50-100 mg/kg) dose-dependently prevented ulcer formation by HCl/ethanol (36-75 % ), absolute ethanol (43-75 %), HCI-ethanol/indomethacin (58-84 %), Pylorus ligation (31-100 %) , cold restraint stress (68-100 %) and histamine (49-100 %). The inhibitory effect at SO and 100 mg/kg against HCl/ethanol was not suppressed by pre-treatment with indomethacin (20 mg/kg, i.p.), TN reduced Shay-ligated gastric acid secretion from 77 mEq/1 in the controls to 46 and 25 mEq/1 for the 50 and 100 mg/kg doses. Augmented histamine-induced gastric acid secretion was reduced from 84 mEq/1 in the controls to 45 and 21 mEq/1 for the two doses of TN, with total inhibition of gastric and duodenal ulcers by the 50 mg/kg dose. Healing rate of chronic acetic acid-induced ulcers was 62 and 83 %, respectively, for the dose of 50 and 100 mg/kg of TN compared with the controls. TN has gastric anti-secretory effects similar to histamine receptor blockers. Its cytoprotective and ulcer healing properties are related to its ability to strengthen gastric mucosal defenses through enhanced gastric mucus production. Key words: Voacanga africana, anti-ulcer activity, gastric secretion, str ess ulcer.
Introduction The discovery in the mid nineteen seventies of anti-secretor y agents capable of low ering the abnormally high acid secretion rates in pat ients marked a turning point in peptic ulcer disease management and treatment (Hall, 1997). Gastric acid hyper secretion th at causes peptic ulcer th rou gh gastric mucosal autodigestion can be of str ess or genetic origin as well as th e result of the interaction of a genetic component with en vironmental factors (Grossma n, 1981 ). The more recent discovery of the role of H elicobacter pylori in the etiology of peptic ulcer disease has led to a radical cha nge in the classical method of treatment and/or management of gastro-du odenal ulcers (Dixon et al., 1996). Thus, the triple th erap y regimen currently recommended for gastric hyper secretors includes a mixture of antacids, antisecretory agents and antibiotics. In Cameroon, antisecretory agents constitute a major
detriment to effective compliance to the triple therapy regimen due to their prohibitive costs. The resultant has been a dramatic 50 % drop in the rate of ho spital con sultations. This situatio n highlights the need to inten sify the search for ant isecreto ry materials from local medicinal sources. We have demonstrat ed the anti-ulcer properties of the bark aqueous and methanol extracts of Voacanga africana in rats (Tan et al., 199 7a , b, c) as well as an antimicrobial action against sta phylococcus aureus . We ha ve also shown a cytoprot ective action of the fru it aqueous extract of the plant. The latter results have prompted us to pursue fur th er work on the fruit extract since it is a plant part, unlik e the bark, whose exploitation would pose less threat to the survival of the species. V. africana ha s been listed in Cameroon as a useful but endangered plant species. This tree plant is 0944-7113/00/07/06-509 $ 15.00/0
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P. V. Tan and B. Nya sse ride, Tween-Zf and Dimeth yl sulphoxide (DMSO) were from Sigma Chemical Co., St Louis, USA.
widely occurring in all of West Africa, as far as Congo and even Tanzania (Bouquet and Debra y, 1974). Although a recent national ethno botanical survey of Cameroonian plants used in ethno medicine cited V. africana as a useful medicinal plant for the treatment of ectopic testis, orchiti s and gonorrhea (Adjanohoun et al., 1996 ), anti-ulcer activity was not listed as one of its medicina l properties. The medicinal uses cited elsewhere include toothache, leprosy, diarrhoea, generalized oedema and convulsions in children (Dalziel, 193 7; Bouquet and Debray, 1974). Previous phyto chemical work cited by Bouquet and Debray (1974) shows that the grain extract contains 1 alkaloid while the bark and leaf extracts contain 23 and 13 alkaloids, respectively. The total alkaloids are known to possess cardiotonic and hypotensive properties (Quevauvillier et al., 1955). The gastric cytoprotective anti-ulcerogenic and ulcer healing actions of TN, a compound obtained from the fruit extract, are therefore novel findings that we now report. The possible modes of action of the compound are discussed in relation to the pathogenic mechanisms of action of the vario us necrotizing agents used.
Animals
Ma le Wistar rats (160-200g) raised in the animal house of the Faculty of Medicine and Biomedical Sciences, University of Yaounde I, were used. The y were fed a standard laboratory diet (S.P.c. Ltd, Bafoussam, Cameroon) and given fresh water ad libitum. Before the experiments, they were starved for 48 h in wire mesh bottom cages to prevent coprophagy but allowed free access to water. Preparation of the anti-ulcer compound
The mature fruits of V. ajricana were harvested in Yaounde in August/September 1998 and sun dried . Voucher specimen No. HNC/1949 (P. Nana: Collector), has been deposited at the National Herbarium, Yaounde. A 1:1 mixture of methylene chloride and methanol (5 litres) was used to extract 5 kg of dried ground powder. The resulting solid (300 g) was fractionated by bioassay-guided procedure to obtain 5 g of a pure active component (TN) . TN was identified by the Meyer and FeCl3 tests as an alka loid NOT containing phenolic groups . The compound has a melting point of 192 -195°C and an [a]D 305 0 [c, 0.1; MeOH]. 50 mg of TN were dissolved in 3 to 5 drops of DMSO and the solution made up to 100 ml using a 0.25 % solution of Tween-20. A 1 % solution of DMSO in 0.25 % Tween-20 was prepared and used as the vehicle control.
Material and methods Material
Sucralfate (Usiphar-F (Compeigne-France), Cimetidine (Smith-Kline & French, 92090 Paris La Defense, France), Indomethacin (Merk Sharp & Dohme, Great Britain) and Ranitidine (Glaxo Wellcome) were purchased from local pharmacies. Histamine dihydrochlo-
Table 1. Effect of TN on gastr ic ulcers induced by HCUethan ol in rats . Treatment Control TN TN Sucralfate
Dose (mg/kg) 50 100 100
N
6 6 6 8
Ulcer index (mean:!:SEM)
Inhibition
5.49 :!: 0.49 3.54 :!: 0.54 * 1.35 :!: 0.64 ** 2.97:!: 0.36 *
(%)
Mucus production (mg)
36 75 50
96.9:!: 102.2:!: 116.5 :!: 48 .0 :!:
7.9 35.5 * 19.9 * 8.1
Statistical significance relative to control, * p < 0.05 ; <. * p < 0.01; N, number of rat s.
Table 2. Effect of TN on gastric ulcers induced by absolute ethan ol in rats . Treatment
Dose (mg/kg)
Control
TN TN Sucralfate
50 100 200
N 6 6 6 8
Ulcer index (mean:!:SEM)
(%)
Mucu s production (mg)
4.88 2.83 1.20 4.49
43 75 8
124 .2:!: 17.6 114.6:!: 16.1 131.8:!: 15.2 " 55.6 :!: 10.7
:!: :!: :!: :!:
0.12 0.89 * 0.81 ** 0.46
Statistical significance relat ive to control, P < 0.05 ; ""p < 0.01 N, number of rats .
Inhibition
Anti-ulcer compound from Yoacanga africana Ulcer preventive tests:
• HCLIethanol-induced gastric mucosal membrane lesions: Gastric muco sal lesions were induced by the method of Hara and Okabe (1985). The test rats were administered the test drug s while the controls received, orall y, 1 ml of the HClJethanol solution (150 mM HCI in 60 % v/v ethanol). The rat s were killed 1 h later using ether and the stomachs remo ved and observed for lesions in the glandular portion. The area covered by each lesion was measured and the lesions were scored as described previously (Tan et al., 199 7a). • Absolute ethanol-induced gastric membrane lesions: Absolute ethanol-induced lesions were provoked using the method described above for the HClIEthanol-induced lesions. The rats received the test drugs or vehicle followed 1 h later by 1 ml of absolute ethanol by oral route. They were also killed using ether and the lesions formed were observed and scored. • HCVEthanol-induced lesions in rats pre-treated with indomethacin: The effect of pre-treatment with indomethacin on the preventive effect of the test drugs on HCllEthanol-induced gastric lesions was studied following a modification of the method described by Sun et al. (1991). All the rats received indomethacin (20 mg/kg) by intraperitoneal route. 1 h later, the test rats received TN or Sucralfate while the controls received 1 ml of vehicle by oral route. Thi s was followed another hour later by oral admini stration of 1 ml of the HClIethanol solution. The rats were then killed 1 h later using ether and the stomachs observed for lesions. • Pylorus -ligated gastric secretion and ulceration: Following a 48 h fast, the test drugs and vehicle were administered orally to the test and control rats, respec-
511
tively, 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 aI., 1996). • Cold restraint stress-induced gastric lesions: Stress-induced gastric lesions were provoked by placing the rats in stress cages and immersing in cold water (3-5 "C) to the level of the xiphoid process for 90 min. The animals received the test products and vehicle 30 min. prior to immersion in cold water. They were killed by a blow to the head and the stomachs opened and observed for lesions in the glandular portion. • Augmented histamine-induced gastric secretion and ulceration: A modified augmented histamine test was used to study the possible mechanism of anti-secretory activity of TN. Control and test animals received vehicle and TN, respectively, 30 min. before pylorus ligation. Repeated hourly injections of histamine dihydrochloride (1 mg/kg, s.c., x 4) were used in combination with the pylorus-ligated ulcer technique to provoke gastric hypersecretion and ulceration. The gastric juice accumulated in the stomach of each animal was collected 1 h after the last histamine injection and assayed for acid content. • Measurement of acid content of gastric juice: Samples of gastric contents (1 rnl) were analyzed for hydrogen ion concentration by pH-metric titration with 0,1 N NaOH solution using a digital pH meter. The acid content was expressed as mliq/l.
Table 3. Effect of TN on gastric ulcers induced by HClIethanol in rats pre-treated with indomethacin. Treatment Control TN TN Sucralfate
Dose (mg/kg) 50 100 100
N
Ulcer index (mean±SEM)
6 6 6 8
5.13 ± 0.14 2.18 ± 0.30 * 0.80 ± 0.50 *,. 6.77 ± 2.40
Inhib ition (%)
Mucus production (mg) 96.0 ± 0.7 118.2 ± 11.8 * 177.4 ± 40.3 " 47.8 ± 9.3
58 84
o
Statistical significance relative to control, *p < 0.05; * "p < 0.01; N , number of rats.
Table 4. Effect of TN on gastric secretion and ulceration in the rats. Treatment
Dose (mg/kg)
Control
TN TN Cimetidine
50 100 50
N
Gastric acidity (rnliq/l ) (mean±SEM)
ulcer index (mean±SEM)
6 6 6 12
77.20 :!: 5.80 47.45 ± 6.72 * 24.91 :!: 4.83"" 72.10:!:11.53
3.15 ± 2.18 ± 0.00 ± 1.10 ±
Statistical significance relative to control,
"p < 0.05; ""p < 0.01; N, number of rats.
0.69 0.68 * 0.00 " '" 0.29 *"
Inhibition (% ) 31 100 68
512
P. V. Tan and B. Nyasse
Ulcer healing test:
• Acetic acid-induced chronic gastric ulceration: The method of Takagi et al. described by Yamamoto et al. (1992) was used. The rat stomachs were exteriorized under light ether anaesthesia and the surface of the anterior wall of the glandular stomach was brought into contact with 1 ml of glacial acetic acid using a cylinder (10 mm in diameter) for 30 sec. The test animal s and controls received the test drugs and vehicle, respectively, by oral route for 10 days starting from the fourth day after the acetic acid treatm ent. All the anim als received food and wat er normally during the treatment period. They were depriv ed of food overnight after the last dose of the treatment and sacrificed the next day using ether. The stomachs were removed and examin ed for ulcers. The length and width of each ulcer was measured and the product (mm-) was taken as the ulcer index. Healing rates were then calculated by comparing test animal ulcer indices with the contro ls. • Measurement of mucus production: Gastric mucus production was measured in the rats subjected to
HCVethanol-, absolute ethanol-, indomethacinIHCIethanol- and acetic acid-induced lesions. The gastric muco sa of each rat was gently scraped using a glass slide and the mucus obtained was weighed using a precision electronic balanc e. The same experimenter performed this operation each time. • Statistical analysis: Values in tables are given as arithmetic mean s ± standard error of the mean (S.E.M .) The significance of differences between mean s was calculated using the student's z-test.
Results Oral administration of TN at the dose of 50 and 100 mg/kg dose-dependently prevented the formation of gastric lesions induced by the HCl/ethanol solution. Ulcer index scores reduced from 5.49 in the controls to 1.35 when 100 mg/kg of TN was used. Sucralfate (100 mg/kg) also inhibited th e formation of gastric mucos al lesions, but it was less effective (49 % inhibition ) than
Table 5. Effects of TN on histamin e-indu ced gastro-duodenal ulcers and gastric secretion in rats. Dose (mg/kg)
Treatm ent Contro l TN TN Ranitidine
25 50 100
N
Gastric acidity (mEqll)
Gastric ulcer (mean±SEM)
Du odenal ulcer (mean±SEM)
Inh ibiti on (%) .
6 6 6 6
84 .37 45.68 20 .83 5.83
1.93 ± 0.22 0.86 ± 0.0 9" 0.00 ± 0.00 " " 0.00 ± 0.00 **
1.40 0.79 0.00 0.00
49$ 100 100
± 14.79 ± 04 .68* ± 02.38** ± 0.36 " *
± 0.4 3 ± 0.4 1* ± 0.00*" ± 0.0 0* "
Stat istical significance relative to contro l, *p < 0.05; "" p < 0.01 ; N , number of rats. $ Value is average of gastr ic and duodenal inhibition.
Table 6. Effect of TN on cold restraint stress-induced gastric lesion s in rats. Treatment
N
Dose (rug/k g)
Co ntro l TN TN Ran itidine
6
6 6
25 50 50
12
ulcer index (mean ± SEM )
Inhibition (%)
3.81 1.24 0.00 0.00
68 100 100
± ±
± ±
0.48 0.22 * 0.00 ** 0.00**
Statistical significance relative to cont rol , "p < 0.05; * *p < 0.01; N, number of rats.
Table 7. Effect of TN on th e healing rate of gastric ulcers induced by acetic acid in rat s. Treatm ent Cont rol TN TN Sucralfate
Do se (mglkg) 50 100 100
N
Ulcer index (mea n±SEM)
Heal ing rate (%)
Mucus production (mg)
6 6 6 6
346 .40 ± 131. 80 ± 57.41 ± 107.04 ±
62 83 69
36.9 1± 3.02 83 .73 ± 7.96 " 104.65 ± 1.96 ** 42.25 ± 2.35
32.93 29 .60 * 19.02 " ** 36. 10*
Statistical significance relative to control, *p < 0.05; " "p < 0.01 ; *"" p < 0.001; N, number of rats.
Anti-ulcer compound from Voacanga africana
513
TN at the same dose. TN promoted higher levels of healing of acetic acid-induced chronic gastric ulcers (62 mucus production than Sucralfate compared with the to 83 % ) following dail y treatment with TN (50 and 100 mg/kg, respectively) for 10 days. A healing rate of controls (Table 1) 69 % was recorded for Sucralfate (100 mg/kg). Unlike The inhibitory effect of TN at 50 and 100 mg/kg against HCl/ethanol was not suppressed by pre-treat- Sucralfate, TN promoted significantly higher levels of ment with indomethacin. This was not the case with mucus production (83 to 104 mg) during the treatment Sucralfate whose preventive effect (49 %) against period compared with the controls (37 mg ). HCI/ethanol disappeared completely following pretreatment with indomethacin (Table 3). Mucus production increased in a dose-dependent manner when inDiscussion creasing doses of TN were given to the rats. Sucralfate promoted lower mucus production compared with the The anti-ulcer methods were selected in order to test TN for gastric cytoprotective and anti-secretory activicontrols following indomethacin pre-treatment. Table 2 shows the results obtained when absolute ty, as well as protection against ulcers of cold stress oriethanol was used as the necrotizing agent. Gastric le- gin. The HCI/ethanol and absolute ethanol methods sions observed were macro morphologically similar to were used to screen the compound for gastric cytoprothose obtained using the HClIethanol solution. Oral tection against the two mucosal irritant substances, administration of TN at doses of 50 and 100 mg/kg and pre-treatment with indomethacin was used to test dose-dependently prevented the formation of gastric le- if cytoprotection was endogenous prostaglandinsions induced by absolute ethanol (% inhibition = 42 linked. Oral administration of TN to the rats preventand 75, respectively). Sucralfate at the dose of 200 ed the formation of gastric lesions induced by mg/kg was not effective aga inst absolute ethanol (% HCI/ethanol, absolute ethanol, indomethacin/HClinhibition =7.9). Neither TN nor Sucralfate significant- ethanol, cold restraint stress, pylorus ligation, and sigly improved mucus production in absolute ethanol- nificantly enhanced the healing of chronic ulcers intreated rats compared with the controls (Table 2). duced using acetic acid. The gastric cytoprotective efTable 4 shows the results obtained after subjecting fect of TN against absolute ethanol suggests that its the rats to pyloric ligature. Lesions produced were ei- protective action in HCI/ethanol-treated model was rether pointed or raised inflammations unlike the lated to an effect other than the simple neutralization stripped lesions caused by absolute ethanol or of the acid in the necrotizing mixture. A proposed HCI/ethanol. TN (50 mg/kg) produced significantly model for the pathogenesis of ethanol-induced gastric lower ulcer index scores (2.18) compared with the con- mucosal damage in rats involves superficial aggressive trols (3.15), and completely inhibited lesion formation cellular necrosis as well as the release of tissue-derived at the dose of 100 mg/kg. Cimetidine (50 mg/kg) also mediators such as histamine and Leucotriene C4 • These reduced ulcer index scores and % inhibition (67.9) was mediators act on gastric microvasculature, triggering a higher than for TN at the same dose. The gastric juice series of events that lead to mucosal and possibly subobtained 6 h after pylorus ligation was highly acidic mucosal tissue damage (Oates and Hakkinen, 1988). (77 mliq/l) in the control rats although the volumes of The cytoprotective action of TN may, therefore, ingastric contents were similar for all the treatments. TN volve a blockade of the generation of these mediators (50 and 100 mg/kg) dose-dependently reduced gastric or their action on microvasculature. Such a blockade of acidity (46 and 25 mEq/l, respectively) unlike Cimeti- histamine would also be responsible for the antisecredine. The augmented histamine-induced gastric acid se- tory activity of TN observed in the pylorus ligated ulcretion was reduced from 84 mEg/1 in the control rats cer model. to 45 and 21 mEg/1 for the rats that received 25 and 50 Treatment with indomethacin causes gastric mucosal mg/kg of TN, respectively. Neither gastric nor duode- injury through a reduction of endogenous prostaglannal ulcers were observed in animals that received TN din (PG) synthesis, inhibition of gastroduodenal bicar(50 mg/kg) or Ranitidine (100 mg/kg) (Table 5 ). bonate secretion, disruption of the mucosal barrier and The effects of subjecting the rats to a combination of reduction of mucosal blood flow (Whittle, 1977; Sellrestraint and cold stress are shown in Table 6. The con- ing et aI., 1987; Flemstrom et aI., 1982; Miller et aI., trol rats had hemorrhagic lesions in the glandular 1982). However, pre-treatment with indomethacin pristomachs 90 min. after cold immersion. TN (25-50 or to HCI/ethanol did not affect the previously obmg/kg) dose-dependently prevented lesion formation, served effect of TN. Similar results have been interpretwith complete inhibition of lesion formation at the ed by Yamamoto et al. (1992) and Sun et al. (1992) to dose of 50 mg/kg. Gastric and duodenal lesions were mean that the cytoprotective action of the compound also absent in rats given 100 mg/kg of Ranitidine. involves a direct mucosal protection similar to that of Table 7 shows a dose-d ependent enhancement of the PGs and not related to endogenous PGs. PGs are
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P. V. Tan and B. Nyasse
known to prevent experimentally-induced ulcers and to protect the gastro-duodenal mucosa against various ulcerogens. Gastric mucosal protection by TN may involve an enhancement of mucus production as was observed in HClIethanol, HClIethanol/indomethacin and acetic acid ulcer models. Sucralfate, a mucosal strengthener known to stimulate mucus, bicarbonate and PG secretion (Rang and Dale, 1991) did not significantly enhance mucus production compared with TN. The ability of TN to inhibit gastric acid secretion was tested using the pylorus ligation method and the possibility that the observed reduction in gastric acidity could be due to a blockade of histamine receptors was tested using the augmented histamine test. Further more, since ulceration due to cold stress is also histamine linked, activity against stress ulcer was tested when the augmented histamine test gave positive results. Shay's pylorus-ligated ulcer technique also demonstrated the anti-ulcer effect of TN, with complete inhibition of ulcer formation at the dose of 100 mg/kg accompanied by a significant reduction in gastric acidity to 25 mEq/l. Similar levels of gastric acidity have previously been observed not to cause gastric rnucosallesions in pylorus ligated rats (Tan et al., 1996). Pepsin and HCI are important for the formation of pylorus-ligated ulcers (Shay et al., 1945). Thus, the significant reduction in gastric acidity suggests that the cytoprotective action of TN may be exercised through an anti-secretory effect similar to that of Hz blockers of histamine, in addition to a possible direct mucosal protection against mucosal autodigestion by pepsin. TN reduced, significantly, the volume and acid concentration of histamine-induced gastric hyperacidity, preventing the formation of both gastric and duodenal ulcers compared with the controls. Such anti-histaminic effects may also explain the total inhibitory action of TN against cold restraint stress-induced ulcer (Senay and Levine, 1967) since mast cells are known to degranulate and release histamine in response to stress (Guth and Hall, 1966). In addition, TN may also act through scavenger activity, on singlet oxygen, the highly reactive mutagenic oxygen species implicated (through lipid peroxydation) in the mechanism of water immersion stress-induced gastric ulcer (Yoshikawa and Miyagawa, cited by Yamamoto et al., 1992). This possibility was, however, not tested in the present work. The ulcer-healing test also showed a dose-dependent enhancement of the healing of chronic ulcers induced using glacial acetic acid. This was associated with improved mucus production, which can protect the rat stomach mucosa against its own peptic secretions and enhance the healing process. Increased gastric mucosal blood flow, and increased oxygen and nutrient supply to the ulcerated area, which have been cited (Svanes et al., 1991; Hirose et al., 1991) as contributing mecha-
nisms to the healing of acetic acid-induced ulcer, may have been implicated in the healing effect of TN but these were not measured. In conclusion, our results suggest that TN taken orally in experimental ulcer models, has cytoprotective, anti-secretory and healing properties which may explain the clinical effect of the fruit extract of V. africana when employed in ethnomedicine for the management of complaints symptomatic of peptic ulcer disease. These effects are likely due to the ability of TN to strengthen gastric mucosal defenses through enhanced mucus production as well as an anti-secretory effect similar to that of Hz receptor antagonists of histamine. TN appears to be a useful compound for the development of new anti-ulcer drugs and its toxicity profile and detailed mechanism of action await further studies. 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, and Grant F/2626-2 (BN).
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Address P. V. Tan, Animal Physiology laboratory, Department of Animal Biology and Physiology, Faculty of Science, P.O. Box 812, University of Yaounde I, Cameroon. e-mail: [email protected]