Antiulcer Properties of the Ethanol Root Extract of Cissampelos mucronata.

ABSTRACT

The study was undertaken to investigate the antiulcer properties of the ethanol root extract of Cissampelos mucronata. A. Rich (Menispermaceae). Three models of experimental ulcer induction in rats were employed in the study. Oral median lethal dose (LD₅₀) was determined in mice using Lorke's method, and the isolated tissue preparations were used to investigate the effect of the extract on calcium ion mobilization in the ileum and H₂-receptor blocking property in nongravid rat uterine strip. Study of the acute toxicity test indicated an LD₅₀ of 288.53 mg/kg. Antiulcer study showed that the extract exhibited dose-dependent antiulcer properties in the order indomethacin > histamine > stress-induced ulcers. The ulcer index recorded for the extract against ulcer induced by indomethacin and histamine were significant at p < 0.05. In vitro. pharmacological studies indicated that the extract possessed ability to block the voltage-gated calcium channels; nevertheless, it did not antagonize histamine-induced relaxation of the rat uterine smooth muscle. The results indicate that the ethanol root extract of C. mucronata. exhibited antiulcer property especially against indomethacin-induced ulcer. Cytoprotection and antispasmodic mechanisms of action may be responsible for the antiulcer properties.

Keywords:

• Antiulcer
• Cissampelos mucronata
• root extract

Introduction

Peptic ulcer disease (PUD) is a common clinical ailment, and it is estimated that 1 out of 10 people will develop an ulcer at some time in their life (Graham, 1999). PUD accounts for approximately 50% of all causes of upper gastrointestinal bleeding (Silverstein et al., 1981). Generally, about 10% of patients presenting with abdominal pain are diagnosed with PUD; prevalence has decreased in developed countries over the past three decades but is increasing in developing countries (Graham, 1999). Despite the liberal use of conventional antiulcer agents, PUD has continued to have a significant morbidity and mortality toll.

Based on the preponderance of evidence demonstrating the efficacy of medicinal plants in the management of ulcers (Al-Harbi et al., 1997; Anandan et al., 1999; Akah et al., 2001; Austin & Jegadeesan, 2002), a lot of attention is being focused on herbal medicine as an alternative in the pharmacotherapy of peptic ulcer disease. This formed the basis for the antiulcer study of the root extract of Cissampelos mucronata. A. Rich (Menispermaceae), used in folklore medicine for the treatment of a variety of stomach problems in eastern parts of Nigeria.

C. mucronata., known among the Igbo tribe of Nigeria as ekenweofia. or obeakaenwe., is a climbing shrub, which is widespread in dry parts of Africa (Hutchison & Dalziel, 1954) and hammocks of Florida and the Everglade keys (Benson, 1957). The genus Cissampelos. is found throughout the Amazon in Peru, Brazil, Ecuador, and Columbia and is cultivated by many people to beautify their gardens; it has 30–40 species of vines (Lewis & Lewis, 1977). The root of the plant is fibrously thickened and tastes bitter with persistent minty taste (Nwafor et al., 2002). The root and bark of C. mucronata. and related species Cissampelos pareira. and Cissampelos oweriansis. have been claimed to be used locally in Nigeria to prevent abortion, in menorrhagia, as a sedative, and in the treatment of stomachaches (Oliver, 1969). The root of C. pareira., referred to as “midwives' herb” by indigenous people in the Amazon, is used in tropical countries to prevent a threatened miscarriage as well as to stop uterine hemorrhages (Lewis & Lewis, 1977). It is also believed to be helpful for menstrual cramps, difficult menstruation, pre- and postnatal pain; the Indians use the decoction of the leaf and stem as an oral analgesic (Lewis & Lewis, 1977). The leaf and root extracts are used as anthelmintic in northern parts of Nigeria (Audu, 1995).

The genus Cissampelos. contains alkaloids characteristic of other members of the family Menispermaceae. Saponins and sterols are common; occasionally, triterpene ethereal oils, polyterpenes, and polyphenols are also present (Bruneton, 1995; Akah & Nwafor, 1999). In other research, the bis.benzylisoquinoline alkaloids have been found to be the anti-inflammatory constituent of related species, C. pareira.. The antiulcer activities of the methanol leaf extract (Akah & Nwafor, 1999) and fractions (Nwafor & Akah, 2003) of C. mucronata. have been documented. The spasmolytic activity of the aqueous leaf extract has been published (Offiah et al., 1996). The ethanol root extract possesses potent uterine smooth muscle relaxant properties (Nwafor et al., 2002), sedative properties (Akah et al., 2002), and spasmolytic potency (Nwafor et al., 2003). Previous phytochemical analysis of the ethanol root extract indicated the presence of the following constituents: flavonoids, sterols/triterpenes, tannins, alkaloids, glycosides, and carbohydrates (Nwafor et al., 2002).

Materials and Methods

Chemicals and drugs

The following chemicals and drugs were used in the experiment: indomethacin (Greenfield International Ltd., London, England), cimetidine (Meridian Drug House, Thane), stilbesterol (Bristol Pharmaceuticals Ltd, London, England), histamine, verapamil (Sigma, St. Louis, MO, USA), glucose (M & B, England), ethanol, )magnesium chloride heptahydrate, sodium chloride (BDH, England, potassium chloride (Hopkin and William, England), sodium carbonate, (Reidel-De Haen Ag, Germany), calcium chloride, sodium hydrogen phosphate, sodium hydrogen carbonate (Merck, Germany), and Tween 85 (Janssen, Belgium).

Animals used

The following animals were used: Swiss albino mice (20–32 g), Swiss albino rats (95–155 g), and guinea pig (300–460 g). All the animals were procured from the Animal House of the Department of Pharmacology and Toxicology, University of Nigeria, Nsukka. They were acclimatized for 5 days with free access to water and food before the commencement of the experiment. The rats and mice were fed with standard pellets (Guinea feeds PLC, Nigeria), and the guinea pig was fed with a local grass, Penicum maximum. L. The animals were maintained under standard 12-h light/dark cycle throughout the duration of the study.

Collection and identification of plant material

Fresh root of C. mucronata. was collected in August from Isuofia, Anambra State, Nigeria, and was botanically identified by Mr. J.M. Ekekwe of the Department of Botany, University of Nigeria, Nsukka. A voucher specimen was deposited in the university herbarium.

Preparation of the extract

The roots were chopped into smaller pieces and air-dried for 10 days. This was further pulverized using a mortar and pestle; 46.30 g was cold-macerated in 250 ml of absolute ethanol for 48 h. The decoction was filtered using filter paper and stored in a refrigerator.

Determination of extractive yield

The extractive yield was determined by taking 1 ml of the filtrate and evaporating it to dryness. Using the weight of the extract in 1 ml of the filtrate, the weight of the entire volume of the extract was calculated and expressed as percentage of the weight of the starting powdered root.

Acute toxicity test

The extract was tested orally for acute toxic effect in mice using the method proposed by Lorke (1983). Nine mice were divided into three groups of three animals per group. Doses of 10, 100, and 1000 mg/kg were administered to the three groups of animals, respectively. The animals were monitored for death within 24 h. Because the three mice that received 1000 mg/kg of the extract died, doses of 140, 225, and 370 mg/kg were subsequently given to three groups of one mouse per group, respectively. These animals were again monitored for mortality for 24 h. The geographic mean of the least dose of the extract that killed the animals and the highest dose that did not kill the animals were taken as the median lethal dose (LD₅₀).

Antiulcer test

The ethanol root extract (EE), suspended in 3% Tween 85, was tested for antiulcer activities using three models of experimental gastric ulcers. These included indomethacin- (Urishidani et al., 1979), histamine- (Langason et al., 1994), and stress-induced ulcers (Langason et al., 1994). Two dose levels of the extract were employed in the study in each of the models. Twenty rats were used for each model. They were divided into four groups of five rats each. Groups 1 and 2 were given 5 ml of 3% Tween 85 and cimetidine 100 mg/kg, respectively, and groups 3 and 4 were administered 50 and 100 mg/kg of the extract, respectively. All administrations were by the oral route. After 30 min, ulcers were induced with the respective agents (indomethacin 30 mg/kg p.o., histamine 2 mg/kg p.o., and cold restrain for stress model). After 8 h (for indomethacin and histamine), the animals were sacrificed and the stomach removed and opened along the greater curvature. The stomach was rinsed under a stream of water and pinned flat on a corkboard. The stomachs were observed with a hand lens (×10). Erosions formed on the glandular portions of the stomach were counted and each given a severity rating on a 1–3 scale based on the diameter of the ulcer (viz., 1,≤1 mm; 2,>1 mm ≤ 2 mm; 3,>2 mm) (Main + Whittle, 1975). The overall total divided by a factor of 10 was designated as the ulcer index (UI) for that stomach.

For stress-induced ulcer, 30 min after administration of drugs, each animal was introduced into a metal restrainer and kept for 18 h in a refrigerator maintained at 10–15°C. The animals were then scarified, their stomachs removed and opened along the greater curvature, and the ulcer index calculated as above. Cimetidine was used for comparison. The percentage ulcer protection was calculated as follows (Suziki et al., 1976):

Effect of the extract on calcium channel in the ileum

The experiments were set up as described previously (Anonymous, 1974). Segments of the guinea pig ileum of about 2 cm long were suspended in an aerated 50 ml organ bath filled with Tyrode solution (deficient in KCl) and maintained at 37 ± 1°C. The preparation was set up under a resting tension of 0.5 g and allowed to equilibrate for 60 min, during which the bathing fluid was changed every 10 min to prevent the accumulation of toxic metabolite. At the end of a 60-min equilibration period, a cumulative dose-response relationship for KCl was established with contact time of minute after each addition. The ileum was then washed repeatedly with the physiological solution and incubated with 20 µg/ml of the extract for 10 min; after which, a second cumulative dose-response curve for similar concentrations of KCl as before was established. When the response to KCl had returned to normal after repeated washings, the procedure was then repeated with 40 µg/ml of the extract. The responses were recorded in triplicate using a two-channel recorder (Gemini 7070, Ugo Basile, Italy) through an isotonic transducer.

Effect of the extract on calcium channel in the rat uterus

The nongravid rat uteri were stimulated into estrus by pretreatment with 0.1 mg/kg of stilbesterol subcutaneously 24 h before use. The isolated tissue preparation was set up using standard procedures (Anonymous, 1974). Following this, the animals were sacrificed by a blow on the head and exsanguinated. The uterus was isolated and each horn cut open longitudinally into a sheet. Each sheet was cut into two strips. Each strip was suspended in a 50-ml organ bath containing high potassium ion, calcium ion-free depolarizing solution, and washed for 30 min with the depolarizing solution to remove intra- and extracellular calcium ions. The depolarizing solution was of the following composition g/l: sodium chloride 1.58, sodium hydrogen carbonate 1.26, potassium chloride 7.46, magnesium chloride hexahydrate 0.25, and glucose 1.98. Similar timing as in the ileum was used. Cumulative concentration-response curve for CaCl₂ was obtained using 30-s contact time for each increment in concentration during the cumulative addition. A second curve was then established in the presence of 10 µg/ml of the extract added 10 min before. Further experiment was performed with 40 µg/ml concentration of the extract when the response had returned to normal after repeated washings. The last cumulative contractile effect of CaCl₂ was determined in the presence of verapamil (1 µg/ml).

Effect on histamine H₂-receptors in rat uterus

Uterine strip preparation was set up as described above but suspended in De-Jalon's solution. The composition of De-Jalon's solution was as follows (g/l): sodium chloride 90, potassium chloride 0.20, calcium chloride 0.20, sodium hydrogen carbonate 1.00, sodium hydrogen phosphate 0.05, magnesium chloride 0.10, and glucose 1.00. The resting tension was 0.5 g, and responses were recorded in triplicate using a two-channel recorder (Gemini 7070, Ugo Basile) through an isotonic transducer.

Statistical analysis

Results were expressed as mean ± standard error of mean. The test results were compared to the control values using Student's t.-test, and the difference was regarded as significant at p < 0.05.

Results

After extraction, the extractive yield was determined to be 3.24% of the starting dry root, while acute toxicity tests indicated a median lethal dose of 288.53 mg/kg. At 50 and 100 mg/kg, the extract exhibited 57% and 78% protection against ulcer induced by indomethacin (Table 1). The reductions in ulcer indices with these two dosage regimens were dose-dependent and significant at p < 0.05. At 100 mg/kg, the ulcer protection offered by the extract (78.74%) was not statistically different from that of cimetidine 100 mg/kg (87.51%), a standard antiulcer agent, indicating comparable efficacy. With respect to histamine-induced ulcer, the percentage ulcer protections were as follows: cimetidine 100 mg/kg (53.86%), extract 100 mg/kg (34.60%), and extract 50 mg/kg (15.46%). Although the protection offered by cimetidine was better than that of extract at the same dose level, the difference was not statistically significant (p < 0.05) (Table 1). In stress-induced ulcer, the extract exhibited better ulcer protective potency than cimetidine when both were administered at 100 mg/kg dose (43.23% and 40.55%, respectively); again, the difference was not statistically significant at 95% confidence interval (Table 1). At 50 mg/kg, the extract produced 35% protection against ulcer induced by stress, indicating a dose-dependent ulcer protective effect.

Table 1.. Effect of the extract on ulcer induced by different ulcerogens (percentage ulcer protection is indicated in parenthesis).

	Ulcer index (percentage ulcer protection in parentheses)
Ulcerogenic agents	Tween 85 (5 ml/kg)	Cimetidine (100 mg/kg)	Extract (50 mg/kg)	Extract (100 mg/kg)
Indomethacin (30 mg/kg p.o)	26.67 ± 1.45	3.33 ± 0.20	11.33 ± 0.35	5.67 ± 0.67
	—	(87.51%)	(57.52%)	(78.74%)
Histamine (2 mg/kg p.o)	8.67 ± 1.77	4.00 ± 0.58	7.33 ± 1.72	5.67 ± 1.29
	—	(53.86%)	(15.46%)	(34.60%)
Stress	12.33 ± 0.67	7.33 ± 2.03	8.00 ± 1.32	7.00 ± 2.75
	—	(40.55%)	(35.12%)	(43.23%)

*Significant p < 0.05; n = 5.

In the isolated guinea pig ileum, the extract neither contracted nor relaxed the tissue preparation. In addition, the extract (20–40 µg/ml) potently inhibited the contraction induced by cumulative addition of potassium chloride (10–160 µg/ml) in guinea pig ileum suspended in potassium ion-free Tyrode solution. This inhibition was found to be concentration-dependent, and at 40 µg/ml, the potassium chloride–induced contraction was virtually abolished (Fig. 1). The extract (10–20 µg/ml) also inhibited calcium chloride–induced (20–320 µg/ml) a contraction of the isolated nongravid rat uterine strip immersed in high potassium ion, calcium ion–deficit depolarizing solution (Fig. 2). The inhibition was also concentration-dependent. Verapamil (1 µg/ml) evoked a much higher inhibition of the calcium chloride–induced contraction. Histamine-induced (4 µg/ml) relaxation of the nongravid rat uterine strip was not antagonized by the extract (20–80 µg/ml); rather, there was slight potentiation of the relaxant effect. In addition to completely blocking the relaxant effect of histamine (4 µg/ml), cimetidine (20 µg/ml) also induced contraction of the isolated tissue preparation of the rate uterus in the presence of histamine (Table 2).

Figure 1 Effect of the extract on contractions induced by cumulative addition of potassium chloride in guinea pig ileum.

Figure 2 Effect of the extract on contractions induced by cumulative addition of calcium chloride in nongravid rat uterine strip.

Table 2.. Effect of the extract on histamine-induced relaxation of nongravid rat uterine strip muscle.

Agent/concentration	Relaxation (cm)
Extract (20 µg/ml)	0.5 ± 0.1
Histamine (4 µg/ml)	1.10 ± 0.2
Extract (20 µg/ml) + histamine (4 µg/ml)	1.50 ± 0.4
Extract (40 µg/ml) + histamine (4 µg/ml)	1.70 ± 0.3
Extract (80 µg/ml) + histamine (4 µg/ml)	1.75 ± 0.6
Cimetidine (20 µg/ml) + histamine (4 µg/ml)	Contraction

n = 5.

Discussion

As a result of the age-long efficacy demonstrated by a wide array of medicinal plants, almost 65% of the world's population have incorporated them into their primary modality of health care (Farnsworth et al., 1985). One of the major priorities in ethnopharmacological research is to study the efficacy of the use of a whole plant or part of it as an herbal remedy. Cissampelos mucronata. is used in the treatment of a variety of ailments (Lewis & Lewis, 1977; Audu, 1995; Nwafor et al., 2002). This study was undertaken to investigate the claim that the alcohol root extract is effective in the management of gastrointestinal dysfunctions, especially peptic ulcer disorder.

The oral median lethal dose (LD₅₀) of 288.53 mg/kg indicates that the ethanol root extract is relatively more toxic than the methanol leaf extract of which the oral LD₅₀ had previously been demonstrated to be 8500 mg/kg (Akah & Nwafor, 1999). Although the extract decreased ulcer indices in the three models of experimental ulcer induction used in the study, significant reduction (p < 0.05) was noted only in indomethacin-induced ulcer. Indomethacin and related nonsteroidal anti-inflammatory drugs (NSAIDs) are known to induce gastrointestinal ulceration by inhibition of the biosynthesis of prostaglandins that are important in gastrointestinal mucosal repair (Banoob, 2002). Consequently, the ability of the extract to significantly protect the animals against ulcer induced by indomethacin signifies possible cytoprotective mechanism of action.

Prostaglandins suppress acid secretion, stimulate gastric mucus and bicarbonate secretion, as well as enhance and maintain mucosal blood flow (Wolfe et al., 1999), while decreased mucosal blood flow leads to increased mucosal breakdown and necrosis (Gottlieb et al., 1986; Gonzalez, 1990). Studies in animals show that stress-induced reductions in mucosal prostaglandin synthesis may render the gastric mucosa more susceptible to acid injury (Wightkin, 1980; Hawkey & Rampton, 1985). Deriving from the protective effect of the extract against indomethacin-induced ulcer, it is postulated that the cytoprotective potency might be responsible for the reductions in ulcer scores noted with the extracts in stress-induced ulcer.

The extract neither contracted nor relaxed the isolated tissue preparation of the guinea pig ileum. This correlates with the results noted earlier with the methanol leaf extract (Akah & Nwafor, 1999) and the ethanol root extract (Nwafor et al., 2003). However, the extract potently inhibited the contraction induced by potassium chloride in guinea pig ileum suspended in K⁺-free Tyrode solution. High K⁺ depolarizes the membrane, opens the voltage-dependent Ca²⁺ channels, increases Ca²⁺ influx, and elicits sustained contraction (Hiraoka et al., 1968; Karaki et al., 1997). Ability to block this type of contraction shows inhibitory activity at the voltage-dependent calcium ion channels. Furthermore, the inhibitory activity of extract against contraction induced by calcium chloride in guinea pig ileum suspended in high potassium ion, Ca²⁺ ion–deficit depolarizing solution further shows inhibition of Ca²⁺ mobilization into the intracellular compartments. In addition, gastrin and acetylcholine stimulate the parietal cell to produce acid by stimulating Ca²⁺-dependent signaling pathway (Hoogerwerf & Pasricha, 2001); and verapamil has been reported to inhibit gastric acid secretion and thereby offer protection against induced ulceration (Ogle et al., 1982; Jain et al., 1994). This underscores the role of calcium mobilization in the control of gastric acidity and might explain the ulcer protective potency of extract. Furthermore, inhibition of Ca²⁺-evoked contractions of K⁺-depolarized tissues is commonly accepted as a test for agents that act nonspecifically by inhibiting Ca²⁺ participation in excitation–contraction-coupling process (Northnover, 1977; Quitana, 1978). The nonspecific activity of the extract has been demonstrated in our previous study in which the extract was shown to antagonize contractions induced by various spasmogens (acetylcholine, serotonin, nicotine, and histamine), abolished spontaneous rhythmic movement of the rabbit jejunum, and potently inhibited gastrointestinal motility (Nwafor et al., 2003). These are typical signs of antispasmodic activity (Offiah et al., 1996), a desirable property of a putative antiulcer agent (Mersereau et al., 1988).

Classical H₂-receptor antagonists are used as standard therapy for peptic ulcer; hence, agents such as cimetidine inhibit acid secretion by H₂-receptor blockade (Feldman & Burton, 1990ab). Because histamine induces relaxation by H₂-histaminergic receptor stimulation (Brown & Roberts, 2001), an in vitro. model of inhibiting histamine-induced relaxation of uterine smooth muscle was adopted as a measure of H₂-receptor antagonistic potency. The extract did not antagonize the relaxant effect of histamine on the uterine strip muscle, indicating lack of histamine H₂-receptor antagonistic property. Cimetidine exhibited a potent histamine H₂-receptor blocking property by exerting complete blockade of the histamine-induced relaxant effect.

Conclusions

The ethanol root extract of Cissampelos mucronata. protected rats against ulcer induced by various ulcerogens especially indomethacin-induced ulcer. The mechanism of the antiulcer activity is postulated to be by cytoprotection and inhibition of calcium ion mobilization into the intracellular compartments. One or two of the phytochemical constituents, previously documented to be present in the ethanol root extract, might be responsible for the activities; further studies will be directed at isolating the bioactive component(s) responsible for the these activities. This study justifies the use of the extract in the treatment of peptic ulcer in some parts of Nigeria.

Acknowledgment

The authors are grateful to the technical staff of the Department of Pharmacology and Toxicology, University of Nigeria, Nsukka, for technical assistance.