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Antinociceptive and smooth muscle contracting activities of the methanolic extract of Cassia tora leaf
Journal of Ethnopharmacology 81 (2002) 205 /209 www.elsevier.com/locate/jethpharm
Antinociceptive and smooth muscle contracting activities of the methanolic extract of Cassia tora leaf F.C. Chidume a,*, H.O. Kwanashie b, J.O. Adekeye c, C. Wambebe a, K.S. Gamaniel a a
Department of Pharmacology and Toxicology, National Institute For Pharmaceutical Research and Development (NIPRD), Idu Industrial Area, P.M.B. 21, Garki, Abuja, Nigeria b Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria, Nigeria c Department of Veterinary Pathology and Microbiology, Ahmadu Bello University, Zaria, Nigeria Received 7 September 2001; received in revised form 1 March 2002; accepted 11 March 2002
Abstract The leaves of Cassia tora Linn. (Family: Caesalpiniaceae) were soxhlet extracted with methanol. The spasmogenic effects of the extract were evaluated on guinea pig ileum, rabbit jejunum and mice intestinal transit. Antinociceptive activity of the extract was also evaluated in the mice. The LD50 values of the extract in mice were /2000 mg/kg i.p. and p.o. The extract contracted smooth muscles of guinea pig ileum and rabbit jejunum in a concentration-dependent manner. Atropine reversibly blocked this activity. Mepyramine also reduced the contractile amplitude due to the extract in a concentration-dependent manner. The extract increased intestinal transit in mice dose dependently. C. tora extract significantly (P B/0.05) reduced the number of acetic acid induced abdominal constrictions in mice and the effect was comparable to that of aspirin (150 mg/kg i.p.). The extract also significantly (P B/ 0.05) reduced the nociceptive response of mice to increased force (g). The effects were dose-dependent. The studies suggest that the use of C. tora , traditionally, as a purgative and in the treatment of other ailments is justifiable. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cassia tora ; Guinea pig ileum; Rabbit jejunum; Intestinal transit; Antinociception
1. Introduction Cassia tora Linn. (Caesalpiniacea) is a shrub that is widely used in traditional medicine in Africa for the treatment of various ailments. The leaves, roots and even the whole plant are employed in the treatment of impetigo, ulcers, helminthiasis and as a purgative (Dalziel, 1955; Irvine, 1961). The powdered leaves are applied to ulcers and to parasitic skin conditions. A fresh leaf decoction may be used as lotion for the same purpose. The decoction from the whole plant or root is taken orally. Also, the fermented leaves are pounded and added to food or ‘local gin’ and taken orally for anthelmintic and purgative effects (Dalziel, 1955; Irvine, 1961; personal communications). The popular use of this plant possibly indicates its value in the alleviation of
* Corresponding author. Tel.: /234-9-523-9089; fax: /234-9-5231043. E-mail address: [email protected] (F.C. Chidume).
the ailments for which they are employed. However, there is the need to establish the scientific basis for which these claims are made. The objective of this study was to evaluate the potential value of the plant as a purgative and analgesic, using laboratory procedures.
2. Materials and methods 2.1. Plant preparation and extraction The shrub, C. tora was collected from Zaria, Kaduna State, Nigeria in January. The plant was authenticated by A. Musa, a plant taxonomist in the Department of Biological Sciences, Faculty of Science, Ahmadu Bello University, Zaria, Kaduna State, Nigeria. A herbarium specimen was deposited and was given a voucher specimen number 1370. The leaves were air-dried for 1 week and were then pulverised in a mortar and sieved to fine powder. The leaf powder was extracted with
0378-8741/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 0 7 9 - X
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F.C. Chidume et al. / Journal of Ethnopharmacology 81 (2002) 205 /209
methanol in a soxhlet extractor for 24 h (Harbone, 1973). The extract was concentrated in water bath and the percentage yield was calculated to be 34% w/w.
concentrations of C. tora extract were tested. The effects of atropine and mepyramine on the tissue responses to the extract were also tested. The median effective concentration of the extract (EC50) was also determined.
2.2. Animals Swiss albino mice (20 /31 g) of both sexes, male rabbits (2.2 /2.5 kg) and male guinea pigs (350 /420 g) were used for the investigations. They were obtained from the Animal House, Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria and from the Animal Facility Centre, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja. The animals were fed ad libitum with standard feed and water except when fasting was needed in the course of the study. 2.3. Drugs and chemicals Aspirin (Sigma, USA), Acetylcholine (Sigma), Histamine (Sigma), Atropine (Sigma), Mepyramine (May and Baker), Carbachol (Sigma), Glacial acetic acid (Searle, Essex, England) and Methanol (B.DH Ltd. Poole, England) were used. 2.4. Pharmacological studies 2.4.1. Acute toxicity study (LD50) The acute toxicity of the extract was estimated in albino mice by the intraperitoneal (i.p.) and oral (p.o.) routes. The procedure involved the modification of the method of Lockes (1983). The method estimates the dose of the extract that will kill 50% of the animal population by the route given. Forty albino mice were starved of feed but allowed access to water 24 h prior to the study. They were then grouped into ten (of four mice each). The first five groups were used for the i.p. study while the remaining groups were used for the oral (p.o.) routes of study. The extract was given at doses of 10, 100, 1000, 1500 and 2000 mg/kg. The mice were then observed for 24 h for behavioural effects such as ataxia, excitement, nervousness, alertness or death. 2.4.2. Studies on guinea pig ileum preparation The adult guinea pig was starved for about 17 h with free access to water. It was then killed by a blow to the head and the abdomen cut open. The ileum was excised and about 2 cm piece was suspended in a 25 ml organ bath containing aerated Tyrode solution maintained at 37 8C. The physiological solution was constituted in millimole (mM) as NaCl 136.8; KCl 2.7; NaH2PO4 0.14; D-Glucose 5.5; NaHCO3 12.0; CaCl2 1.3; MgCl2 0.5 dissolved in 1 litre of distilled water. The set up was connected to microdynamometer recorder set at sensitivity of 1.5 and speed of 24 mm/min. At equilibrium, the effect of acetylcholine, histamine and different
2.4.3. Studies on isolated rabbit jejunum The adult rabbit was starved of feed for about 18 h and was killed by a blow on the head. About 2 cm piece jejunum was excised from the opened abdomen and suspended in a 25 ml organ bath containing aerated Tyrode solution maintained at 37 8C. The effect of acetylcholine, the leaf extract, atropine and mepyramine were studied on the piece of jejunum. The responses were recorded on a microdynamometer set at sensitivity of 1.5 and speed of 24 mm/min. The median effective concentration of the extract (EC50) was also determined. 2.4.4. Studies on mice intestinal transit The study was carried out according to the method of Akah et al. (1998). Albino mice of either sex (20 /24 g) were used for the study. The mice were starved for 24 h prior to the experiment but were allowed free access to water. The mice were then grouped into five (of four mice each). The first group received normal saline (10 ml/kg p.o.) and served as the control. The second, third and fourth groups had oral administration of extracts at doses of 100, 200 and 400 mg/kg respectively, while the fifth group received carbachol (1 mg/kg i.p) and served as the reference standard. Thirty minute after the treatments, 0.5 ml of a 5% charcoal suspension in 10% tragacanth powder was administered orally to each animal. The animals were sacrificed 30 min later and the abdomen cut open. The percentage of distance travelled by the charcoal plug in the small intestine (from the pylorus to the caecum) was determined for both the treated and control groups (Akah, 1989). 2.4.5. Antinociceptive activity The antinociceptive effect of the methanolic extract of C. tora leaf was tested in mice using acetic acid-induced abdominal constriction and force-induced tail flick methods as follows. 2.4.5.1. Abdominal constriction test. The method of Siegmund et al. (1957) as modified by Koster et al. (1959) was adopted. Two doses of the extract (200 and 400 mg/kg) were administered intraperitoneally to different groups of four mice each. 30, 60, 90 and 120 min after administration of the extract, 0.75% glacial acetic acid was administered intraperitoneally to all the mice at the dose of 10 ml/kg. Five minute after injection of acetic acid, the number of abdominal constrictions made within 5 min by each mouse were counted with the aid of a counter. Similar tests were done using aspirin (150 mg/kg i.p.) as reference standard and normal saline for the control.
F.C. Chidume et al. / Journal of Ethnopharmacology 81 (2002) 205 /209
2.4.5.2. Tail flick test. The modified techniques of Takagi et al. (1966) and Huong et al. (1996) were followed. The screening technique involved the use of Ugo Basile Analgesymeter (Cat. Number 7200) for the evaluation of the force-induced stimulus. This instrument exerts a force that increases at a constant rate. The force was applied to the mouse tail, which was placed on a small plinth under a cone-shaped pusher with a round tip. The scale was read off at the point when the mouse suddenly withdrew its tail and that marked the point at which the mouse felt the force. Prior to treatment, the nociceptive response was taken for each mouse in order to establish the baseline values. Two doses of the extract (50 and 100 mg/kg i.p.) were administered to two groups (of four mice each) and aspirin (150 mg/kg i.p.) was used as a reference standard. The group with normal saline served as the control. The test was then repeated and the nociceptive response was measured every 30 min over a 120 min observation period. 2.4.6. Statistical analysis The results were expressed as mean9/SEM and significance of difference between the control and treated groups were determined using one-way analysis of variance (ANOVA). P -values B/0.05 were taken to be statistically significant. Two-way ANOVA was used to analyse the analgesic studies. Dunnets and Bonferrari tests were adopted for the post hoc analyses to the oneway and two-way ANOVA, respectively.
3. Results
207
dent contraction of the isolated rabbit jejunum. The EC50 of the extract on the isolated rabbit jejunum was 0.09 mg/ml. Atropine (0.04 mg/ml) blocked the contractile effect of the extract on rabbit jejunum. 3.4. Studies on mice intestinal transit The leaf extract of C. tora had no effect on mice intestinal transit up to the dose of 200 mg/kg. However, the dose of 400 mg/kg significantly increased the transit time (P B/0.05). The effect of 400 mg/kg dose was comparable to that of carbachol (1 mg/kg i.p.; Table 1). 3.5. Antinociceptive activity 3.5.1. Abdominal constriction test C. tora leaf extract (200 /400 mg/kg i.p.) significantly (P B/0.05) reduced the intensity of acetic acid-induced abdominal constrictions in mice. The effect was comparable to that of aspirin (150 mg/kg i.p.). The maximal antinociceptive effects were observed at the 60th and 120th min for the 200 and 400 mg/kg doses, respectively (Fig. 1). 3.5.2. Tail flick test The leaf extract of C. tora (50 /100 mg/kg i.p.) significantly (P B/0.05) increased the ability of the treated mice to withstand heavier force when compared with the control. The effect was also comparable to that of aspirin (150 mg/kg i.p.). The maximal antinociceptive effect was observed 30 min following administration of the extract (Table 2).
3.1. Acute toxicity study (LD50) 4. Discussion At 2000 mg/kg i.p. and p.o., no remarkable adverse effects such as ataxia, excitement, nervousness or even death were seen during the observation period of 24 h. 3.2. Studies on guinea pig ileum preparation The leaf extract of C. tora (0.04 /25.6 mg/ml) caused a concentration-dependent contraction of the isolated guinea pig ileum as did acetylcholine (E/0.04 mg/ml) and histamine (E/0.04 mg/ml). The EC50 of the extract on guinea pig was 1.33 mg/ml. Mepyramine (0.8/3.2 mg/ ml) reduced the contractile effect of C. tora extract (1.6 mg/ml) on the guinea pig ileum in a concentrationdependent manner. Atropine (0.04 mg/ml) completely blocked the contractile effect of C. tora extract (0.8 mg/ ml) on the isolated guinea pig ileum. 3.3. Studies on isolated rabbit jejunum The leaf extract of C. tora (0.04 /2.56 mg/ml) caused a transient relaxation followed by a concentration depen-
The acute toxicity (LD50), spasmogenic and antinociceptive effects of the leaf extracts of C. tora were evaluated in this study. The LD50 values for the leaf extract were estimated to be ]/2000 mg/kg for both oral and i.p. routes of administration suggesting that the extract was relatively safe. LD50 value /1 g are generally considered safe for all practical purposes in the laboratory (Lockes, 1983). This finding supports the wide application of the plant in traditional medicines. The contractile effects of the extract on both guinea pig ileum and rabbit jejunum, as well as the increase in mice intestinal transit also agree with the use of the plant locally as purgative. Some standard purgatives are known to bring about their effects by increasing gastrointestinal motility, causing decreased absorption of salt and water secondary to decreased transit time (Brunton, 1990). Purgative effects will lead to enhanced gastric and intestinal emptying which is desirable in the treatment of worm infestation. Indeed, most of the effective anthelmintics currently in use also have purga-
F.C. Chidume et al. / Journal of Ethnopharmacology 81 (2002) 205 /209
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Table 1 Effect of methanolic leaf extract of C. tora on mice intestinal transit Treatment group Mean intestinal length (cm) Mean distance travelled by charcoal (cm) Movement of charcoal as percentage of intestinal length Norma l saline 10 ml/kg p.o.
35.7390.6
15.9791.5
44.70
C. tora (p.o. ) 100 mg/kg 200 mg/kg 400 mg/kg
34.8591.8 34.6790.8 33.3391.8
18.0092.4 18.5091.9 24.3393.2
51.65 53.36 73.00*
Carbachol (i.p. ) 1 mg/kg
35.7391.0
27.8096.4
77.81*
*, Significant (P B 0.05) increase in motility one-way ANOVA.
tive components for worm expulsion. This finding, therefore, strongly supports the use of the plant as anthelmintic in traditional medicine. The antagonism between the extract and atropine on both guinea pig ileum and rabbit jejunum suggests that muscarinic cholinergic mechanism may be involved in the activities of the extract. Cholinomimetics are known to stimulate the smooth muscles of the gut through the muscarinic receptors and this stimulatory effect is blocked by atropine. The effects of the extract were also blocked by mepyramine, suggesting that the extract may interfere with histaminergic mechanisms via the H1-receptors
to cause contraction of smooth muscles of the gut. The results also revealed that the extract produced significant antinociceptive responses that were dose and time dependent in mice. The results were comparable to those of aspirin. Although the mechanism of action of the extract in relation to antinociception is not revealed by the present study, antinociceptive property may be of added advantage to the use of the extract against various ailments. In conclusion, the methanolic extract of C. tora leaf has demonstrated gastrointestinal stimulatory and antinociceptive effects, which seem to be in conformity with
Fig. 1. Effect of the methanolic extract of C. tora (200 /400 mg/kg i.p.) on glacial acetic acid / induced abdominal constriction in mice. * / significant reduction of abdominal constriction (P B/0.05).
F.C. Chidume et al. / Journal of Ethnopharmacology 81 (2002) 205 /209 Table 2 Effect of the methanolic extract of C. tora leaf (50 /100 mg/kg i.p.) on force-induced noriception in mice Tail flick force (g)9SEM Drug (mg/ kg) Control
30 min
146.2592.3
60 min
157.5091.8
90 min
120 min
150.0093.1
153.7599.4
C. tora 50 100
207.75*92.4 206.25*91.9 195.00*93.7 262.50*95.2 217.50*92.7 195.0093.1
195.0093.2 187.5092.9
Aspirin 150
232.50*94.8 225.00*92.3 217.50*93.9 213.75*96.0
*, Significant (P B 0.05) increase in force borne; two-way ANOVA.
the ethnomedical uses of the plant. There is a potential for the development of a herbal purgative and analgesic. Further studies will be pursued on the chronic toxicity and anti-inflammatory activities.
Acknowledgements The authors of this paper are grateful to Charles Ebute and David Akumka for their technical assistance. They are also indebted to Charles Balogun for the secretarial work.
209
References Akah, P.A. 1989. Purgative Potentials of Euphorbia heterophylla . Fitoterapia 60, 45 /48. Akah, P.A., Orisakwe, O.E., Gamaniel, K.S., Shittu, A. 1998. Evaluation of Nigerian Traditional Medicines; 11 Effects of some Nigerian folk remedies on peptic ulcer. Journal of Ethnopharmacology 62, 123 /127. Brunton, L.L. 1990. Agents affecting gastrointestinal water flux and motility, Digestants and Bile acids. In: Gilman, G.A., Rall, T.W., Nies, A.S., Taylor, P. (Eds.), The Pharmacological Basis of Therapeutics, eight ed.. Permamon Press, pp. 914 /932. Dalziel, J.M. 1955. The Useful Plants of West Tropical Africa. Crown Agents, London, pp. 260 /265. Harbone, J.B. 1973. A guide to Modern techniques of Plant AnalysisPhytochemical Methods, first ed.. Chapmand and Hall, New York. Huong, N.T.T., Matsumoto, K., Yamasaki, K., Duc, N.M., Nham, N.T., Watanabe, H. 1996. Effects of Vietnamese Ginseng on Opioid agonist and conditional fear stress-induced antinociception. Phytomedicine 3, 33 /39. Irvine, F.R. 1961. Woody Plants of Ghana. Oxford University Press, London, pp. 291 /293. Koster, R., Anderson, N., Debber, E.J. 1959. Acetic acid for analgesic screening. Federation Proceedings 18, 412. Lockes, D. 1983. A new approach to acute toxicity testing. Archives of Toxicology 54, 275 /287. Siegmund, E.A., Cadmus, R.A., Lu, G. 1957. Screening of analgesic including aspirin type compound based upon the antagonism of chemically induced writhing in mice. Journal of Pharmacology and Experimental Therapeutics 119, 184 /186. Takagi, H., Inukai, T., Nakama, M. 1966. A modification of Haffner’s method for testing analgesics. Japanese Journal of Pharmacology 16, 287 /294.
Antinociceptive and smooth muscle contracting activities of the methanolic extract of Cassia tora leaf F.C. Chidume a,*, H.O. Kwanashie b, J.O. Adekeye c, C. Wambebe a, K.S. Gamaniel a a
Department of Pharmacology and Toxicology, National Institute For Pharmaceutical Research and Development (NIPRD), Idu Industrial Area, P.M.B. 21, Garki, Abuja, Nigeria b Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria, Nigeria c Department of Veterinary Pathology and Microbiology, Ahmadu Bello University, Zaria, Nigeria Received 7 September 2001; received in revised form 1 March 2002; accepted 11 March 2002
Abstract The leaves of Cassia tora Linn. (Family: Caesalpiniaceae) were soxhlet extracted with methanol. The spasmogenic effects of the extract were evaluated on guinea pig ileum, rabbit jejunum and mice intestinal transit. Antinociceptive activity of the extract was also evaluated in the mice. The LD50 values of the extract in mice were /2000 mg/kg i.p. and p.o. The extract contracted smooth muscles of guinea pig ileum and rabbit jejunum in a concentration-dependent manner. Atropine reversibly blocked this activity. Mepyramine also reduced the contractile amplitude due to the extract in a concentration-dependent manner. The extract increased intestinal transit in mice dose dependently. C. tora extract significantly (P B/0.05) reduced the number of acetic acid induced abdominal constrictions in mice and the effect was comparable to that of aspirin (150 mg/kg i.p.). The extract also significantly (P B/ 0.05) reduced the nociceptive response of mice to increased force (g). The effects were dose-dependent. The studies suggest that the use of C. tora , traditionally, as a purgative and in the treatment of other ailments is justifiable. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cassia tora ; Guinea pig ileum; Rabbit jejunum; Intestinal transit; Antinociception
1. Introduction Cassia tora Linn. (Caesalpiniacea) is a shrub that is widely used in traditional medicine in Africa for the treatment of various ailments. The leaves, roots and even the whole plant are employed in the treatment of impetigo, ulcers, helminthiasis and as a purgative (Dalziel, 1955; Irvine, 1961). The powdered leaves are applied to ulcers and to parasitic skin conditions. A fresh leaf decoction may be used as lotion for the same purpose. The decoction from the whole plant or root is taken orally. Also, the fermented leaves are pounded and added to food or ‘local gin’ and taken orally for anthelmintic and purgative effects (Dalziel, 1955; Irvine, 1961; personal communications). The popular use of this plant possibly indicates its value in the alleviation of
* Corresponding author. Tel.: /234-9-523-9089; fax: /234-9-5231043. E-mail address: [email protected] (F.C. Chidume).
the ailments for which they are employed. However, there is the need to establish the scientific basis for which these claims are made. The objective of this study was to evaluate the potential value of the plant as a purgative and analgesic, using laboratory procedures.
2. Materials and methods 2.1. Plant preparation and extraction The shrub, C. tora was collected from Zaria, Kaduna State, Nigeria in January. The plant was authenticated by A. Musa, a plant taxonomist in the Department of Biological Sciences, Faculty of Science, Ahmadu Bello University, Zaria, Kaduna State, Nigeria. A herbarium specimen was deposited and was given a voucher specimen number 1370. The leaves were air-dried for 1 week and were then pulverised in a mortar and sieved to fine powder. The leaf powder was extracted with
0378-8741/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 2 ) 0 0 0 7 9 - X
206
F.C. Chidume et al. / Journal of Ethnopharmacology 81 (2002) 205 /209
methanol in a soxhlet extractor for 24 h (Harbone, 1973). The extract was concentrated in water bath and the percentage yield was calculated to be 34% w/w.
concentrations of C. tora extract were tested. The effects of atropine and mepyramine on the tissue responses to the extract were also tested. The median effective concentration of the extract (EC50) was also determined.
2.2. Animals Swiss albino mice (20 /31 g) of both sexes, male rabbits (2.2 /2.5 kg) and male guinea pigs (350 /420 g) were used for the investigations. They were obtained from the Animal House, Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria and from the Animal Facility Centre, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja. The animals were fed ad libitum with standard feed and water except when fasting was needed in the course of the study. 2.3. Drugs and chemicals Aspirin (Sigma, USA), Acetylcholine (Sigma), Histamine (Sigma), Atropine (Sigma), Mepyramine (May and Baker), Carbachol (Sigma), Glacial acetic acid (Searle, Essex, England) and Methanol (B.DH Ltd. Poole, England) were used. 2.4. Pharmacological studies 2.4.1. Acute toxicity study (LD50) The acute toxicity of the extract was estimated in albino mice by the intraperitoneal (i.p.) and oral (p.o.) routes. The procedure involved the modification of the method of Lockes (1983). The method estimates the dose of the extract that will kill 50% of the animal population by the route given. Forty albino mice were starved of feed but allowed access to water 24 h prior to the study. They were then grouped into ten (of four mice each). The first five groups were used for the i.p. study while the remaining groups were used for the oral (p.o.) routes of study. The extract was given at doses of 10, 100, 1000, 1500 and 2000 mg/kg. The mice were then observed for 24 h for behavioural effects such as ataxia, excitement, nervousness, alertness or death. 2.4.2. Studies on guinea pig ileum preparation The adult guinea pig was starved for about 17 h with free access to water. It was then killed by a blow to the head and the abdomen cut open. The ileum was excised and about 2 cm piece was suspended in a 25 ml organ bath containing aerated Tyrode solution maintained at 37 8C. The physiological solution was constituted in millimole (mM) as NaCl 136.8; KCl 2.7; NaH2PO4 0.14; D-Glucose 5.5; NaHCO3 12.0; CaCl2 1.3; MgCl2 0.5 dissolved in 1 litre of distilled water. The set up was connected to microdynamometer recorder set at sensitivity of 1.5 and speed of 24 mm/min. At equilibrium, the effect of acetylcholine, histamine and different
2.4.3. Studies on isolated rabbit jejunum The adult rabbit was starved of feed for about 18 h and was killed by a blow on the head. About 2 cm piece jejunum was excised from the opened abdomen and suspended in a 25 ml organ bath containing aerated Tyrode solution maintained at 37 8C. The effect of acetylcholine, the leaf extract, atropine and mepyramine were studied on the piece of jejunum. The responses were recorded on a microdynamometer set at sensitivity of 1.5 and speed of 24 mm/min. The median effective concentration of the extract (EC50) was also determined. 2.4.4. Studies on mice intestinal transit The study was carried out according to the method of Akah et al. (1998). Albino mice of either sex (20 /24 g) were used for the study. The mice were starved for 24 h prior to the experiment but were allowed free access to water. The mice were then grouped into five (of four mice each). The first group received normal saline (10 ml/kg p.o.) and served as the control. The second, third and fourth groups had oral administration of extracts at doses of 100, 200 and 400 mg/kg respectively, while the fifth group received carbachol (1 mg/kg i.p) and served as the reference standard. Thirty minute after the treatments, 0.5 ml of a 5% charcoal suspension in 10% tragacanth powder was administered orally to each animal. The animals were sacrificed 30 min later and the abdomen cut open. The percentage of distance travelled by the charcoal plug in the small intestine (from the pylorus to the caecum) was determined for both the treated and control groups (Akah, 1989). 2.4.5. Antinociceptive activity The antinociceptive effect of the methanolic extract of C. tora leaf was tested in mice using acetic acid-induced abdominal constriction and force-induced tail flick methods as follows. 2.4.5.1. Abdominal constriction test. The method of Siegmund et al. (1957) as modified by Koster et al. (1959) was adopted. Two doses of the extract (200 and 400 mg/kg) were administered intraperitoneally to different groups of four mice each. 30, 60, 90 and 120 min after administration of the extract, 0.75% glacial acetic acid was administered intraperitoneally to all the mice at the dose of 10 ml/kg. Five minute after injection of acetic acid, the number of abdominal constrictions made within 5 min by each mouse were counted with the aid of a counter. Similar tests were done using aspirin (150 mg/kg i.p.) as reference standard and normal saline for the control.
F.C. Chidume et al. / Journal of Ethnopharmacology 81 (2002) 205 /209
2.4.5.2. Tail flick test. The modified techniques of Takagi et al. (1966) and Huong et al. (1996) were followed. The screening technique involved the use of Ugo Basile Analgesymeter (Cat. Number 7200) for the evaluation of the force-induced stimulus. This instrument exerts a force that increases at a constant rate. The force was applied to the mouse tail, which was placed on a small plinth under a cone-shaped pusher with a round tip. The scale was read off at the point when the mouse suddenly withdrew its tail and that marked the point at which the mouse felt the force. Prior to treatment, the nociceptive response was taken for each mouse in order to establish the baseline values. Two doses of the extract (50 and 100 mg/kg i.p.) were administered to two groups (of four mice each) and aspirin (150 mg/kg i.p.) was used as a reference standard. The group with normal saline served as the control. The test was then repeated and the nociceptive response was measured every 30 min over a 120 min observation period. 2.4.6. Statistical analysis The results were expressed as mean9/SEM and significance of difference between the control and treated groups were determined using one-way analysis of variance (ANOVA). P -values B/0.05 were taken to be statistically significant. Two-way ANOVA was used to analyse the analgesic studies. Dunnets and Bonferrari tests were adopted for the post hoc analyses to the oneway and two-way ANOVA, respectively.
3. Results
207
dent contraction of the isolated rabbit jejunum. The EC50 of the extract on the isolated rabbit jejunum was 0.09 mg/ml. Atropine (0.04 mg/ml) blocked the contractile effect of the extract on rabbit jejunum. 3.4. Studies on mice intestinal transit The leaf extract of C. tora had no effect on mice intestinal transit up to the dose of 200 mg/kg. However, the dose of 400 mg/kg significantly increased the transit time (P B/0.05). The effect of 400 mg/kg dose was comparable to that of carbachol (1 mg/kg i.p.; Table 1). 3.5. Antinociceptive activity 3.5.1. Abdominal constriction test C. tora leaf extract (200 /400 mg/kg i.p.) significantly (P B/0.05) reduced the intensity of acetic acid-induced abdominal constrictions in mice. The effect was comparable to that of aspirin (150 mg/kg i.p.). The maximal antinociceptive effects were observed at the 60th and 120th min for the 200 and 400 mg/kg doses, respectively (Fig. 1). 3.5.2. Tail flick test The leaf extract of C. tora (50 /100 mg/kg i.p.) significantly (P B/0.05) increased the ability of the treated mice to withstand heavier force when compared with the control. The effect was also comparable to that of aspirin (150 mg/kg i.p.). The maximal antinociceptive effect was observed 30 min following administration of the extract (Table 2).
3.1. Acute toxicity study (LD50) 4. Discussion At 2000 mg/kg i.p. and p.o., no remarkable adverse effects such as ataxia, excitement, nervousness or even death were seen during the observation period of 24 h. 3.2. Studies on guinea pig ileum preparation The leaf extract of C. tora (0.04 /25.6 mg/ml) caused a concentration-dependent contraction of the isolated guinea pig ileum as did acetylcholine (E/0.04 mg/ml) and histamine (E/0.04 mg/ml). The EC50 of the extract on guinea pig was 1.33 mg/ml. Mepyramine (0.8/3.2 mg/ ml) reduced the contractile effect of C. tora extract (1.6 mg/ml) on the guinea pig ileum in a concentrationdependent manner. Atropine (0.04 mg/ml) completely blocked the contractile effect of C. tora extract (0.8 mg/ ml) on the isolated guinea pig ileum. 3.3. Studies on isolated rabbit jejunum The leaf extract of C. tora (0.04 /2.56 mg/ml) caused a transient relaxation followed by a concentration depen-
The acute toxicity (LD50), spasmogenic and antinociceptive effects of the leaf extracts of C. tora were evaluated in this study. The LD50 values for the leaf extract were estimated to be ]/2000 mg/kg for both oral and i.p. routes of administration suggesting that the extract was relatively safe. LD50 value /1 g are generally considered safe for all practical purposes in the laboratory (Lockes, 1983). This finding supports the wide application of the plant in traditional medicines. The contractile effects of the extract on both guinea pig ileum and rabbit jejunum, as well as the increase in mice intestinal transit also agree with the use of the plant locally as purgative. Some standard purgatives are known to bring about their effects by increasing gastrointestinal motility, causing decreased absorption of salt and water secondary to decreased transit time (Brunton, 1990). Purgative effects will lead to enhanced gastric and intestinal emptying which is desirable in the treatment of worm infestation. Indeed, most of the effective anthelmintics currently in use also have purga-
F.C. Chidume et al. / Journal of Ethnopharmacology 81 (2002) 205 /209
208
Table 1 Effect of methanolic leaf extract of C. tora on mice intestinal transit Treatment group Mean intestinal length (cm) Mean distance travelled by charcoal (cm) Movement of charcoal as percentage of intestinal length Norma l saline 10 ml/kg p.o.
35.7390.6
15.9791.5
44.70
C. tora (p.o. ) 100 mg/kg 200 mg/kg 400 mg/kg
34.8591.8 34.6790.8 33.3391.8
18.0092.4 18.5091.9 24.3393.2
51.65 53.36 73.00*
Carbachol (i.p. ) 1 mg/kg
35.7391.0
27.8096.4
77.81*
*, Significant (P B 0.05) increase in motility one-way ANOVA.
tive components for worm expulsion. This finding, therefore, strongly supports the use of the plant as anthelmintic in traditional medicine. The antagonism between the extract and atropine on both guinea pig ileum and rabbit jejunum suggests that muscarinic cholinergic mechanism may be involved in the activities of the extract. Cholinomimetics are known to stimulate the smooth muscles of the gut through the muscarinic receptors and this stimulatory effect is blocked by atropine. The effects of the extract were also blocked by mepyramine, suggesting that the extract may interfere with histaminergic mechanisms via the H1-receptors
to cause contraction of smooth muscles of the gut. The results also revealed that the extract produced significant antinociceptive responses that were dose and time dependent in mice. The results were comparable to those of aspirin. Although the mechanism of action of the extract in relation to antinociception is not revealed by the present study, antinociceptive property may be of added advantage to the use of the extract against various ailments. In conclusion, the methanolic extract of C. tora leaf has demonstrated gastrointestinal stimulatory and antinociceptive effects, which seem to be in conformity with
Fig. 1. Effect of the methanolic extract of C. tora (200 /400 mg/kg i.p.) on glacial acetic acid / induced abdominal constriction in mice. * / significant reduction of abdominal constriction (P B/0.05).
F.C. Chidume et al. / Journal of Ethnopharmacology 81 (2002) 205 /209 Table 2 Effect of the methanolic extract of C. tora leaf (50 /100 mg/kg i.p.) on force-induced noriception in mice Tail flick force (g)9SEM Drug (mg/ kg) Control
30 min
146.2592.3
60 min
157.5091.8
90 min
120 min
150.0093.1
153.7599.4
C. tora 50 100
207.75*92.4 206.25*91.9 195.00*93.7 262.50*95.2 217.50*92.7 195.0093.1
195.0093.2 187.5092.9
Aspirin 150
232.50*94.8 225.00*92.3 217.50*93.9 213.75*96.0
*, Significant (P B 0.05) increase in force borne; two-way ANOVA.
the ethnomedical uses of the plant. There is a potential for the development of a herbal purgative and analgesic. Further studies will be pursued on the chronic toxicity and anti-inflammatory activities.
Acknowledgements The authors of this paper are grateful to Charles Ebute and David Akumka for their technical assistance. They are also indebted to Charles Balogun for the secretarial work.
209
References Akah, P.A. 1989. Purgative Potentials of Euphorbia heterophylla . Fitoterapia 60, 45 /48. Akah, P.A., Orisakwe, O.E., Gamaniel, K.S., Shittu, A. 1998. Evaluation of Nigerian Traditional Medicines; 11 Effects of some Nigerian folk remedies on peptic ulcer. Journal of Ethnopharmacology 62, 123 /127. Brunton, L.L. 1990. Agents affecting gastrointestinal water flux and motility, Digestants and Bile acids. In: Gilman, G.A., Rall, T.W., Nies, A.S., Taylor, P. (Eds.), The Pharmacological Basis of Therapeutics, eight ed.. Permamon Press, pp. 914 /932. Dalziel, J.M. 1955. The Useful Plants of West Tropical Africa. Crown Agents, London, pp. 260 /265. Harbone, J.B. 1973. A guide to Modern techniques of Plant AnalysisPhytochemical Methods, first ed.. Chapmand and Hall, New York. Huong, N.T.T., Matsumoto, K., Yamasaki, K., Duc, N.M., Nham, N.T., Watanabe, H. 1996. Effects of Vietnamese Ginseng on Opioid agonist and conditional fear stress-induced antinociception. Phytomedicine 3, 33 /39. Irvine, F.R. 1961. Woody Plants of Ghana. Oxford University Press, London, pp. 291 /293. Koster, R., Anderson, N., Debber, E.J. 1959. Acetic acid for analgesic screening. Federation Proceedings 18, 412. Lockes, D. 1983. A new approach to acute toxicity testing. Archives of Toxicology 54, 275 /287. Siegmund, E.A., Cadmus, R.A., Lu, G. 1957. Screening of analgesic including aspirin type compound based upon the antagonism of chemically induced writhing in mice. Journal of Pharmacology and Experimental Therapeutics 119, 184 /186. Takagi, H., Inukai, T., Nakama, M. 1966. A modification of Haffner’s method for testing analgesics. Japanese Journal of Pharmacology 16, 287 /294.