Antiulcer Effects of the Aqueous and Organic Extracts of the Stem Bark of Anthocleista vogelii. in Rats

Abstract

The aqueous, hexane, acetone, and methanol extracts of the stem bark of Anthocleista vogelii. Planch. (Loganiaceae) were screened for antiulcerogenic activity using HCl/ethanol gastric necrotizing solution. The methanol extract showed 100% inhibition at a dose of 500 mg/kg compared with the aqueous, hexane, and acetone extracts, which produced 87.91%, 61.39%, and 76.89%, respectively. The methanol and aqueous extracts were further tested against ulceration induced by indomethacin (30 mg/kg) and pylorus ligation at doses of 125, 250, and 500 mg/kg. The aqueous extract at a dose of 500 mg/kg significantly (p ≤ 0.01) reduced indomethacin-induced gastric lesions by 78.62% while the methanol extract at the same dose completely inhibited the action of indomethacin. The aqueous extract of A. vogelii. at doses of 250 and 500 mg/kg induced a decrease of about 15% and 40%, respectively, of the surface area in the pylorus-ligated rats. Similarly, the methanol extract at doses 250 and 500 mg/kg produced a decrease of almost 25% and 70%, respectively. The gastric juice secretion increased significantly (p ≤ 0.05) at a dose of 500 mg/kg in the aqueous extract while the gastric acidity significantly (p ≤ 0.01) decreased at doses of 125 and 500 mg/kg in aqueous and methanol extracts as compared with the control group. These findings show that the aqueous and organic extracts of the stem bark of Anthocleista vogelii. possess potent antiulcer properties thereby lending support to the ethnomedical uses of the plant in the treatment of stomachache.

Keywords:

• Anthocleista vogelii
• antiulcer activity
• Loganiaceae

Introduction

Gastric hyperacidity and ulcer are very common ailments causing human suffering today. Ulcer results from the imbalance between damaging factors within the lumen and protective mechanisms within the gastroduodenal mucosa (Rao et al., 2004). Most of the antiulcer drugs available are thought to act by either neutralising acid secretion or by blocking H₂ receptors, protron pump or by acting as anticholinergics Different therapeutic agents, including herbal preparations, are used to inhibit gastric acid secretion or to boost mucosal defense mechanisms by increasing mucus production (Bafna & Balaraman, 2004).

Anthocleista vogelii. Planch. (Loganiaceae) is a small tree, that grows in the tropical rainforest areas of West Africa (Dalziel, 1955). In African traditional medicine, the stem bark and leaves are used as an anti-inflammatory, antidiabetic, and also in the treatment of wounds (Dalziel, 1955; Irvine, 1961).

Previous studies on the genus Anthocleista. and on this plant showed the presence of alkaloids, xanthones, seco-iridoids, terpenes, and pthalides (Chapelle, 1976; Okorie, 1976). Some information obtained from local traditional healers indicated the use of the stem bark for curing fever, stomachache, and as a purgative. The current study was undertaken to determine the antiulcer potential of the aqueous and organic extracts of the stem bark of Anthocleista vogelii. using three experimental gastric ulcer methods, namely HCl/ethanol induction, indomethacin induction, and pylorus-ligation induction.

Materials and Methods

Animals

The experiments were carried out on Wistar male adult rats, aged between 12 and 16 weeks and weighing between 170 and 200 g. The rats were raised in the animal house of the Faculty of Science of University of Dschang and fed normal laboratory rat diet with food and water given ad libitum.. All animals used were starved for 48 h prior to use.

Collection and preparation of plant material

The stem bark of Anthocleista vogelii. was collected in Bandjoun, in the West Province of Cameroon, during the month of May in 2002. Mr. Paul Mesili, a retired botanist of the Cameroon herbarium, Yaoundé, carried out the authentication. A voucher specimen (BUD 0636) has been deposited at the Botany Department, University of Dschang. The collected fresh stem bark was sun-dried and ground into fine powder. The powder was used for the extraction of potential antiulcerogenic principles into differents solvents (water, hexane, acetone, and methanol).

Extraction of the aqueous plant material

Powdered Anthocleista vogelii. stembark (200 g) was boiled in 2 l of distilled water for 15 min. The decoction was taken and allowed to cool for 30 min at room temperature (24 ± 2°C). The decoction was filtered twice and evaporated to dryness in an air oven at 50°C, to give 20.8 g of the aqueous extract corresponding with an extraction yield of 10.4%.

Extraction of the organic plant material

Three thousand grams of the stem bark powder was soaked with 5 l of hexane for 72 h. The filtrate was concentrated to dryness in a rotary evaporator under reduced pressure at a temperature of 70°C to give 31.6 g of hexane extract (1.05% yield). The original material was then macerated in acetone for 72 h, and the filtrate obtained was concentrated at a temperature of 55°C to give 112.3 g of acetone extract (3.74% yield). The original plant material was further macerated in methanol for 72 h and the filtrate concentrated at a temperature of 65°C to give 676 g of methanol extract (22.53% yield). The aqueous and methanol extracts were soluble in distilled water while the hexane and acetone extracts were solubilized in 3% DMSO.

Chemicals

Indomethacin, DMSO, HCl/ethanol, and NaOH were obtained from Sigma Chemicals (Perth, Western Australia), and cimetidine (Tagamet) and sucralfate (Ulcar) were obtained from a local pharmacy.

Experimental

HCl/ethanol–induced ulcers

Gastric mucosal lesions were induced in male rats using the HCl/ethanol method as described by Hara and Okabe (1985). The animals were divided into 15 groups each consisting of 6 rats. Groups 1 and 2 represented the negative control groups, which received 1 ml/100 g body weight distilled water and 3% DMSO solutions. Groups 3, 4, and 5 represent the positive control groups, which received 1 ml/100 g body weight cimetidine (12 mg/kg) and sucralfate (50 and 100 mg/kg) solutions, respectively. Groups 6 to 15 received also 1 ml/100 g body weight aqueous extract (125, 250, and 500 mg/kg),hexane extract (250 and 500 mg/kg), acetone extract (250 and 500 mg/kg), and methanol extract (125, 250, and 500 mg/kg) solutions of the stem bark of A. vogelii., respectively. All test substances were administered orally.

One hour after drug treatment, 1 ml per 150 g body weight of the necrotizing solution (150 mM HCl in 60% ethanol) was given per os. to each rat. The rats were sacrificed 1 h later using an overdose of ether and the stomach removed and observed for ulcers in the glandular region. The surface area of each lesion was measured and scored as described by Tan et al. (1996). The ulcer index for each rat was taken as the mean ulcer score. The percentage-ulcerated surface was calculated as the total area covered by all lesions expressed as a proportion of the total corpus mucosal surface area. The percentage of inhibition (% I) was calculated using the following formula:

Indomethacin-induced ulcers

In order to ascertain whether the antiulcer properties of the aqueous and methanol extracts were mediated by the stimulation of cyclooxygenase activity, the indomethacin-induced model was utilized as described by Shay et al. (1945). Forty-eight male rats divided into 8 groups of 6 rats each were treated with increasing doses of aqueous and methanol extracts ranging from 125 to 500 mg/kg given orally (p.o.) each as a single dose. The negative control group received distilled water, whereas the positive control received sucralfate at 50 mg/kg body weight. The animals received 1 ml/100 g body weight distilled water, sucralfate, or plant extracts.

One hour after drug administration, each animal received orally 30 mg/kg of indomethacin. All animals were sacrificed five hours later by overdose of ether. The stomach was removed and examined for lesions in the glandular portion. The ulcers produced were scored using a modification of the method described by Tan et al. (1996): 0, absence of ulcer; 1, vessel dilatation and pointed ulcers; 2.5, small ulcers < 4 mm long; 3.5, 4 mm long ≤ ulcers ≥ 5 mm long; 5, large ulcers > 5 mm long. The ulcer index and the percentage of inhibition were estimated as described above.

Pylorus-ligated ulcer technique

This method was used to investigate the neutralizing properties and/or the antisecretory effects of the extracts of A. vogelii.. Fifty-four male rats divided into 9 groups of 6 rats each were given (1 ml/100 g body weight) the aqueous and methanol extracts (125, 250, and 500 mg/kg), cimetidine (12 mg/kg), Maalox (50 mg/kg) or distilled water, respectively.

One hour before the experiment, the pylorus of each rat was tied under light ether anesthesia and the abdominal incision was closed. Six hours after the ligation, the animals were sacrificed using overdose of ether and the stomach removed. A small nick was made at the junction of pylorus ligation and greater curvature. The contents of the stomach were collected in centrifuge tubes. The tubes were centrifuged at 3000 rpm for 10 min. The volume of supernatant fluid was measured and used for pH analysis. The stomach was opened along the greater curvature and ulcers produced were graded according to a modification of the method described by Ganguly (1969): 0, absence of ulcer; 1, vessel dilatation and pointed ulcers; 2.5, small ulcers < 4 mm long; 3.5, 4 mm long ≤ ulcers ≥ 5 mm long; 5, large ulcers > 5 mm long. The ulcer index and the percentage of inhibition were estimated as described above.

Measurement of gastric acidity

Total centrifuged gastric contents (1 ml) from each pylorus ligated rat was analyzed for hydrogen ion concentration by titrating against a 0.01 N solution of NaOH using a pH meter (Suntex TS-2, Taipei, Traiwan). The experiment was done in triplicate.

Measurement of mucus production

Gastric mucus production was measured in the rats subjected to HCl/ethanol, indomethacin, and pylorus ligation–induced lesions according to the method described by Tan and Nyasse (2000). The gastric mucosa of each rat was gently scraped using a glass slide, and the mucus obtained was carfully weighed using a sensitive digital electronic balance. The same experimenter performed this operation each time.

Statistical analysis

Statistical analysis was performed using ANOVA and t.-test, and significance of difference between treatments was accepted at p ≤ 0.05. Data are expressed as mean ± standard error of the mean.

Results

pH of the extracts solutions

As presented in Table 1, the aqueous and methanol extracts of the stem bark of A. vogelii. at the concentration of 50 mg/ml show a respective pH of 4.13 and 3.11.

Table 1 pH of the extract solutions.

Extract solution	pH of the extract solution
Aqueous extract (50 mg/ml)	4.13
Methanol extract (50 mg/ml)	3.11

HCl/ethanol–induced ulcer

The oral administration of the HCl/ethanol solution produced characteristic lesions in the glandular portion of the control rat stomach with a total surface area of 132.61 ± 24.70 and 125.50 ± 7.44 mm², respectively, for groups receiving distilled water and DMSO (3%). The aqueous extract of A. vogelii. produced a dose-dependent inhibition of gastric ulceration ranging from 37.89% at a dose of 125 mg/kg to 87.91% at a dose of 500 mg/kg, with respective ulcer surface areas of 82.37 ± 11.53 and 16.04 ± 5.44 mm². The methanol extract produced 72.53% inhibition at a dose of 125 mg/kg, and there was complete prevention of lesion formation with a dose of 500 mg/kg. The corresponding ulcer surface area was 36.43 ± 10.29 and 0.00 ± 0.00 mm². The hexane and acetone extracts of A. vogelii. (250 and 500 mg/kg) significantly (p < 0.05) prevented gastric mucosal lesions produced by the HCl/ethanol solution (59.20–75.56% I).Animals treated with cimetidine at a dose of 12 mg/kg produced an ulcer surface area of 92.82 ± 16.62 mm² corresponding with 29.84% inhibition, and sucralfate produced a significant (p ≤ 0.05) decrease in ulcer surface area from 132.61 ± 24.70 mm² to 36.87 ± 25.14 mm² at a dose of 50 mg/kg and 25.21 ± 4.26 mm² at a dose of 100 mg/kg, leading to inhibition percentage of 72.20% and 80.99%, respectively. The mean ulcer index scored decreased significantly (p ≤ 0.05) from 5.49 in negative control group to 3.42 and 3.08 in aqueous extract, 3.39 and 0.00 in methanol extract for rats receiving 250 and 500 mg/kg, respectively (Table 2).

Table 2 Effects of the stem bark aqueous and organic extracts of Anthocleista vogelii. on HCl/ethanol–induced gastric lesions in rats.

Treatment	n	Dose (mg/kg)	US area (mm^2)	Ulcer index	% US	% I	Mucus wet weight(mg)
Distilled water	6	—	132.61 ± 24.70	5.49 ± 0.98	22.02	—	51 ± 0.46
DMSO (3%)	6	—	125.50 ± 7.44	5.01 ± 0.50	20.89	—	51 ± 0.48
Cimetidine	6	12	92.82 ± 16.62	5.57 ± 0.76	13.78	29.84	61 ± 0.83
Sucralfate	6	50	36.87 ± 25.14^b.	3.46 ± 1.75	04.82	72.20	78 ± 0.96^b.
	6	100	25.21 ± 4.26^b.	3.72 ± 0.38^a.	03.80	80.99	85 ± 1.20^b.
Aqueous extract	6	125	82.37 ± 11.53^b.	5.09 ± 0.46	12.96	37.89	61 ± 0.77
	6	250	32.79 ± 15.40^b.	3.42 ± 0.37^a.	04.25	75.27	95 ± 0.64^b.
	6	500	16.04 ± 5.44^b.	3.08 ± 0.56^b.	02.58	87.91	96 ± 0.74^b.
Hexane extract	6	250	49.05 ± 10.36^b.	4.18 ± 0.64	07.02	60.92	77 ± 0.82
	6	500	51.20 ± 14.90^a.	4.24 ± 0.61	07.49	59.20	81 ± 1.27
Acetone extract	6	250	46.45 ± 13.49^b.	3.58 ± 0.36	05.74	62.99	92 ± 0.83
	6	500	30.67 ± 13.70^b.	3.93 ± 1.00	03.40	75.56	99 ± 0.78
Methanol extract	6	125	36.43 ± 10.29^b.	4.18 ± 0.45	06.28	72.53	58 ± 0.48^a.
	6	250	19.88 ± 6.17^b.	3.39 ± 0.18^b.	02.79	85.00	96 ± 0.98^b.
	6	500	0.00 ± 0.00^b.	0.00 ± 0.00^b.	00.00	100	100 ± 0.60^b.

n, number of rats per group; I, inhibition; US, ulcer surface.

^a.p ≤ 0.05

^b.p ≤ 0.01, statistically significant relative to control.

Indomethacin-induced gastric lesions

Table 3 shows the results obtained when the aqueous and methanol extracts of the stem bark of A. vogelii. were used to prevent the formation of gastric lesions induced by indomethacin. The total surface of ulceration obtained with controls was 82.97±5.08 mm². The aqueous extract of the stem bark of A. vogelii. induced a significant (p ≤ 0.01) decrease in ulcer surface area from 82.97±5.08 to 39.21±13.34 and 17.74±3.04 mm² at doses of 250 and 500 mg/kg, leading to inhibition percentage of 52.74% and 78.62%, and the methanol extract at a dose of 500 mg/kg completely prevented gastric lesion induced by indomethacin. Sucralfate produced a significant (p ≤ 0.01) reduction in ulcer surface area from 82.97±5.08 mm² to 14.75±3.39 mm² at a dose of 50 mg/kg, leading to inhibition of 82.22%. The mucus weight of the control animals (57 mg) increased to 64 mg in animals treated with the aqueous extract (500 mg/kg) and to 67 and 89 mg in those treated with the methanol extract at doses of 250 and 500 mg/kg, respectively.

Table 3 Effects of the stem bark aqueous and methanol extracts of Anthocleista vogelii. on gastric lesions induced by indomethacin.

Treatment	n	Dose(mg/kg)	US area (mm^2)	Ulcer index	% US	% I	Mucus wet weight(mg)
Control	6	—	82.97 ± 5.08	3.64 ± 0.68	13.96	—	57 ± 0.64
Sucralfate	6	50	14.75 ± 3.39^b.	3.16 ± 0.61	02.43	82.22	59 ± 0.40
Aqueous extract	6	125	70.09 ± 13.83	3.63 ± 0.53	09.82	16.41	52 ± 0.35
	6	250	39.21 ± 13.34^b.	3.22 ± 0.53	06.43	52.74	55 ± 0.97
	6	500	17.74 ± 3.04^b.	3.22 ± 0.33	03.06	78.62	64 ± 0.54
Methanol extract	6	125	50.92 ± 12.66^b.	3.62 ± 0.53	07.50	38.62	56 ± 0.34
	6	250	22.33 ± 9.25^b.	2.63 ± 0.49^a.	03.66	73.09	67 ± 0.53
	6	500	0.00 ± 0.00^b.	0.00 ± 0.00^b.	00.00	100	89 ± 0.10

n, number of rats per group; I, inhibition; US, ulcer surface.

^a.p ≤ 0.05

^b.p ≤ 0.01, statistically significant relative to control.

Pylorus ligation–induced gastric lesions

The results obtained when ulceration of the gastric mucosa was provoked by pylorus ligation are shown in Table 4. Pyloric ligation of rats in the control group produced pointed lesions or raised inflammation. The total surface of ulceration obtained was 64.63±5.24 mm². The aqueous and methanol extracts of A. vogelii. at doses of 250 and 500 mg/kg produced a significant (p ≤ 0.05) decrease in ulcer surface area from 64.63±5.24 mm² in the control group to 53.74±5.65 and 39.04±4.99 mm² for the aqueous extract and 47.68±2.93 and 19.40±3.74 mm² for the methanol extract, respectively. The volume of the gastric juice in the control group (5.71 ml) significantly (p ≤ 0.05) increased to 6.35 ml when the aqueous extract at a dose of 500 mg/kg was administered. Gastric acidity was significantly (p ≤ 0.01) decreased from 134.17±2.04 meq/l in the control group to 101.07±17.35 and 95.48±8.36 meq/l when aqueous and methanol extracts were given at a dose of 500 mg/kg.The mucus weight of the control group (56 g) significantly (p ≤ 0.05) increased to 70 and 82 when the aqueous and methanol extracts were administered at a dose of 500 mg/kg.

Table 4 Effects of the stem bark aqueous and methanol extracts of Anthocleista vogelii. on pylorus ligation ulceration.

Treatment	n	Dose (mg/kg)	US area (mm^2)	Ulcer index	% US	% I	Gastric juice (ml)	Gastric pH	Gastric acidity (meq/l)	Mucus wet weight (mg)
Control	6	—	64.63 ± 5.24	3.91 ± 1.16	16.62	—	5.71 ± 0.71	1.83 ± 0.66	134.17 ± 2.04	56 ± 0.69
Cimetidine	6	12	41.45 ± 8.43^b.	2.33 ± 0.34^a.	06.52	35.87	5.53 ± 0.79	1.58 ± 0.93	89.95 ± 19.71^b.	63 ± 0.60
Maalox	6	50	24.15 ± 2.88^b.	2.72 ± 0.28	03.71	62.63	6.30 ± 0.63^b.	0.97 ± 0.34^a.	115.55 ± 26.13	64 ± 0.66
Aqueous extract	6	125	64.66 ± 6.02	2.86 ± 0.32	11.87	0.00	4.87 ± 1.46	1.17 ± 0.17	105.90 ± 7.40^b.	53 ± 0.42
	6	250	53.74 ± 5.65^a.	2.95 ± 0.32	09.47	16.85	5.13 ± 0.36	1.23 ± 0.31	104.03 ± 35.48	56 ± 0.58
	6	500	39.04 ± 4.99^b.	2.92 ± 0.56	07.50	39.59	6.35 ± 0.63^a.	1.31 ± 0.09	101.07 ± 17.35^b.	70 ± 0.10^a.
Methanol extract	6	125	55.39 ± 8.66	3.30 ± 0.55	09.62	14.30	5.71 ± 0.67	1.19 ± 0.24	115.45 ± 12.58^b.	50 ± 0.34
	6	250	47.68 ± 2.93^b.	3.50 ± 0.44	09.39	26.23	3.73 ± 0.83	1.65 ± 0.55	100.50 ± 36.70	68 ± 0.35^a.
	6	500	19.40 ± 3.74^b.	3.11 ± 0.99	06.40	69.98	4.03 ± 2.30	3.43 ± 1.99	95.48 ± 8.36 ^b.	82 ± 0.78^b.

n, number of rats per group; I, inhibition; US, ulcer surface.

^a.p ≤ 0.05

^b.p ≤ 0.01, statistically significant relative to control.

Discussion and Conclusion

The results of this study show that the aqueous and methanol extracts of the stem bark of A. vogelii. exert a protective action against HCl/ethanol, indomethacin, or pylorus ligation–induced gastric mucosal damage.

The HCl/ethanol method of inducing gastric lesions is a rapid and convenient way of screening plant extracts for antiulcer potency and cytoprotection, which is assessed in terms of the absence or reduction in macroscopically visible lesions (Shay et al., 1945; Hara Okabe, 1985; Tan et al., 2000).

The gastric mucosal protection against HCl/ethanol can be mediated by a number of mechanisms that include enhanced gastric mucosal defense through increased mucus and/or bicarbonate production, reducing the volume of gastric acid secretion or by simply neutralizing the gastric acidity (Antonio et al., 2004). It is evident that this method of inducing ulcers is unable to indicate the exact mechanism of action of the plant extracts.

To probe the possible mechanisms of action of the extracts, their antiulcer potency was tested against indomethacin-induced ulcers. The results obtained show that the ulcer surface area and the mean ulcer index were significantly (p < 0.05) reduced, while the mucus weight increased in the aqueous and methanol extract groups compared with the control group. It is known that indomethacin is a prostaglandin inhibitor that suppresses gastroduodenal bicarbonate secretion, disrupts the mucosal barrier, and reduces endogenous prostaglandin biosynthesis as well as gastric mucosal blood flow in animals (Whittle, 1977; Flemstrom et al., 1982; Selling et al., 1987; Tan et al., 2000). On the other hand, prostaglandins synthesized in large quantities by the gastrointestinal mucosa are known to prevent experimentally induced ulcers caused by various ulcerogens. Thus, when the ulcer lesions are induced by indomethacin, the cytoprotective effect of the plant extracts (the antiulcer agents under investigation) can be mediated through endogenous prostaglandins (Yamamoto et al., 1992).

In an attempt to show the effectiveness in preventing gastric ulcer and also assess the antisecretory activity of the aqueous and methanol extracts of A. vogelii., antiulcer properties were tested against pylorus ligation–induced gastric lesions. The aqueous and methanol extracts of A. vogeli. significantly (p < 0.05) reduced gastric lesions as well as the total acid secreted, while the mucus weight significantly (p < 0.05) increased. Pylorus ligation–induced ulcers are due to autodigestion of the gastric mucosa and breakdown of the gastric mucosal barrier (Sairam et al., 2002). This mechanism points to the fact that the cytoprotective effect of the plant extract might be due to the enhancement of the mucosal barrier through the increased production of prostaglandins. These results also reveal that the aqueous and methanol extracts of the stem bark of A. vogelii. may possess antisecretory potency but should be devoid of a neutralizing effect because the solutions were of very low pH.

Phytochemical studies of the methanol extract of A. vogelii. have revealed the presence of xanthones. Some xanthones, such as jacareubin (JR) and 6-desoxyjacareubin (DJR), have been shown to possess antiulcer activity in rats (Gopalakrishnan et al., 1980), and these compounds may be responsible for the antiulcer activity of A. vogelii. methanol extract.

This study reveals that the aqueous and methanol extracts of the stem bark of A. vogelii. are potent inhibitors of gastric mucosal lesions caused by HCl/ethanol, indomethacin, and pylorus ligation in rats. The cytoprotective action may be mediated by endogenous prostaglandin and inhibition of acid secretion. A study of the action of 1-hydroxy-3,7,8-trimethoxyxanthone isolated from the methanol extract on experimentally induced ulcers is warranted.

Acknowledgments

The authors acknowledge with thanks, the financial assistance of IPICS SWEDEN through the project CAM 02.