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Possible involvement of cholinergic and adrenergic mechanisms in changing contractility of guinea pig ileum by Ipomoea carnea
Journal of Ethnopharmacology 71 (2000) 253 – 259 www.elsevier.com/locate/jethpharm
Possible involvement of cholinergic and adrenergic mechanisms in changing contractility of guinea pig ileum by Ipomoea carnea S.K. Hore *, Ram Ottalwar, K.M. Koley, A.K. Pathak Department of Pharmacology and Toxicology, College of Veterinary Science and A.H., I.G.A.U., Anjora, Durg-491001 (MP), India Received 12 February 1999; received in revised form 4 January 2000; accepted 14 January 2000
Abstract The study aimed to elucidate the mechanism (s) of action of Ipomoea carnea leaf juice (ILJ) in changing contractility of guinea pig ileum. ILJ produced dose-dependent (10 – 10 000 mg/ml) triphasic responses. The initial contractile phase was blocked by atropine (1 mg/ml) but had additive effect with acetylcholine (2 ng/ml) or carbachol (2 ng/ml). Neostigmine (30 ng/ml) or lignocaine (50 mg/ml) failed to alter the response. In cold-induced denervated preparations, this phase was augmented. The relaxatory phase of ILJ was not modified by phenoxybenzamine (35 mg/ml) but was reduced by propranolol (1 mg/ml) and abolished by lignocaine (50 mg/ml). The final contractile phase of ILJ was not affected by atropine (1 mg/ml). These results suggest that the triphasic response of ILJ is possibly mediated through cholinergic, adrenergic and non-cholinergic mechanisms, respectively. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ipomoea carnea leaf juice; Guinea pig ileum; Triphasic response; Cholinergic; Adrenergic; Non-cholinergic
1. Introduction The plant Ipomoea carnea, Jacq. syn. Argyreia superbiens, Cave (Convolvulaceae), besharam in hindi, is found throughout India. Normally, animals refuse to take the plant, because it is not a palatable forage for them (Gahlot and Gupta, * Corresponding author. Present address: Department of Pharmacology and Toxicology, College of Veterinary Science, G.B. Pant University of Agriculture and Technology, Pantnagar-263 145 (U.P.), India.
1984). But in scarcity, like draught condition, they ingest the plant and thereby get poisoned (Shrivastava and Soni, 1987). Both fresh and dry leaves and stems of the plant are equally toxic to animals (Damir et al., 1987). Goats and sheep followed by cattle are the most common livestock species affected (Adam et al., 1973; Gahlot and Gupta, 1984). Beside other signs, the digestive disorders exhibited in the form of ruminal hypermotility and diarrhoea are the common symptoms of poisoning (Gahlot and Gupta, 1984; Tirkey et al., 1987;
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Srilatha et al., 1993). The mechanism (s) involved in altering the gastrointestinal tract contractility by I. carnea is not yet clear. The present study has, therefore, aimed to investigate the mechanism (s) of action of I. carnea leaf juice (ILJ) to produce hypermotility of gut.
32°C and continuously bubbled with air. The preparations were allowed to equilibrate under a resting tension of 0.5 g for 60 min, during which the bathing solution was changed every 15 min. The isotonic contractions (magnification × 8) were recorded on a slow moving (0.025 mm/s) smoked Kymograph paper using a frontal writing lever.
2. Materials and methods
2.5. Elucidation of mechanism(s) of action of ILJ 2.1. Plant material Mature green leaves, collected in the month of March–April from the College campus, were identified and authenticated by Dr D. Karkun, Head of Botany Department, College of Science, Arts and Commerce, Durg, MP, India.
2.2. Preparation of juice One hundred grams of fresh leaves was triturated with 100 ml of distilled water and the juice was centrifuged at 3000 rpm for 15 min. The supernatant was referred to as I. carnea leaf juice (ILJ) and was used for this experiment. The w/w yield of prepared extract was 12.3% in terms of starting crude material. The concentration of extract in the supernatant was 10.4 mg/ml.
2.3. Animals Adult guinea pigs of either sex (300 – 350 g) obtained from the Laboratory Animal Unit of the Department were used in the study. They were maintained under controlled conditions with a standard pellet diet and water ad libitum.
2.4. Preparation of guinea pig ileum The animals were sacrificed by a sharp blow on the head and cutting the neck blood vessels (Ghosh, 1984). The terminal segments (2 cm long) of ileum after discarding 10 cm from the ileocaecal junction, were immediately removed and cleaned. The tissues were then mounted in a 20 ml organ bath containing Ringer – Locke solution (composition in mM: NaCl 154, KCl 5.6, CaCl2 2.2, NaHCO3 6.0 and glucose 11.1) maintained at
To elucidate the mechanism(s) of action of ILJ in guinea pig ileum the steps given below were followed. Each tissue preparation was exposed to only one agonist or one combination. 1. Dose-dependent contractions induced by ILJ (10–10 000 mg/ml), acetylcholine (0.3–1000 ng/ml) and carbachol (1–1000 ng/ml) were recorded. 2. Atropine sulphate (1 mg/ml) was added to the bath fluid 15 min before recording the response of ILJ (25 mg/ml), acetylcholine (ACh, 2 ng/ml) or carbachol (2 ng/ml) to antagonize their effects. 3. The response of ILJ (25 mg/ml), ACh (2 ng/ml) and adrenaline (0.3 mg/ml) were taken before and after incubating the tissue with lignocaine (50 mg/ml) for 5 min. 4. The combined effect of ILJ (25mg/ml) and ACh (2 ng/ml) were observed. 5. The ACh (2 ng/ml) and ILJ (25 mg/ml)-induced contractions were recorded in absence and presence of neostigmine (30 ng/ml, 15 min). 6. The effects of carbachol (2 ng/ml) were seen either alone or in combination with ILJ (25 mg/ml) or ACh (2 ng/ml). 7. Effect of isoprenaline on acetylcholine (1 mg/ ml)-induced contractions was recorded. 8. The ILJ (25 mg/ml) and adrenaline (0.3mg/ml)mediated responses were observed before and after incubating the preparations with either phenoxybenzamine (35 mg/ml, 15 min) or propranolol (1 mg/ml, 10 min) in absence or presence of atropine (1 mg/ml). 9. Several experiments were conducted on the preparations that had been stored at low temperature of 4°C for 96 h for denervation (Lum
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et al., 1966), but were later maintained at 32°C bath temperature during experiment.
2.6. Drugs Acetylcholine chloride (Sigma), L-adrenaline (Loba Chemie), atropine sulphate (Loba Chemie), carbachol (Sigma), isoprenaline hydrochloride (Sigma), lignocaine hydrochloride (Astra-IDL), neostigmine methylsulphate (Tablets India), Phenoxybenzamine hydrochloride (SKF), and propranolol hydrochloride (I.C.I.) were used in the experiment.
2.7. Statistical analysis The experimental data were analysed by the Student’s t-test for obtaining the level of significance. PB 0.05 was considered as significant. ED50 values were determined by regression analysis, using the least square methods and were expressed as mean with 95% confidence limits (Snedecor and Cochran, 1967).
3. Results I. carnea leaf juice (ILJ, 25 – 100 mg/ml) produced either triphasic (83.3%) or biphasic (16.7%) responses in guinea pig ileum. In the former group of tissues, the triphasic response consisted
Fig. 2. Dose-contraction curves of (a) acetylcholine (ACh) and carbachol (CB); and (b) I. carnea leaf juice (ILJ) in respect to percent maximum response of ACh in guinea pig ileum (n = 6).
of a fast contraction followed by a brief relaxation and then a sustained contcentration (Fig. 1). A dose-dependent contraction followed by relaxation was produced by ILJ. The dose-response curves (n= 6) of contractile effects of ACh, carbachol and ILJ and relaxatory effect of ILJ have been presented in Figs. 2 and 3. The ED50 values and the contractions required to produce maximum response (Emax) of the above drugs have been listed in Table 1. Fig. 1. (a) Triphasic response of I. carnea leaf juice (, 25 mg/ml) on guinea pig ileum. (b) Blockade of initial contractile phase of I. carnea leaf juice (, 25 mg/ml) by atropine ( , 1 mg/ml). W, wash.
3.1. Initial contractile phase of ILJ Carbachol (2 ng/ml), ACh (2 ng/ml) and ILJ (25 mg/ml) contracted the guinea pig ileum by
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Fig. 4. I. carnea leaf juice (I, 25 mg/ml)-induced contractions in absence and presence of lignocaine (L, 50 mg/ml), neostigmine (N, 30 ng/ml) and propranolol (P, 1 mg/ml) in guinea pig ileum (n =6). *PB 0.05.
Fig. 3. Dose-relaxation curves of (a) isoprenaline (ISP) to acetylcholine (ACh, 1 mg/ml)-induced contraction; and (b) I. carnea leaf juice (ILJ) in guinea pig ileum (n= 6).
29.59 2.96, 24.17 9 1.49 and 15.3 9 1.63 mm, respectively (Fig. 6), which were about 16.7, 13.8 and 9.1% of their respective maximum responses. These effects were completely blocked by atropine (1 mg/ml). The combination of carbachol (2 ng/ ml) and ACh (2 ng/ml), carbachol (2 ng/ml) and ILJ (25 mg/ml), or ACh (2 ng/ml) and ILJ (25 mg/ml) produced additive responses (52.33 93.61,
44.179 2.14 or 40.509 1.18 mm, respectively, Fig. 6, n= 6). Lignocaine (50 mg/ml) failed to block the contractile responses of both ILJ (25 mg/ml, Fig. 4) and ACh (2 ng/ml, Fig. 5), but significantly blocked ILJ (25 mg/ml)-induced relaxation phase (Fig. 8). Though neostigmine (30 ng/ml, 15 min) significantly increased the ACh (2 ng/ml)-induced contraction from 26.169 1.05 to 70.8393.47 mm (n= 6, Fig. 5), but failed to alter the response of ILJ (25 mg/ml, Fig. 4). The preand post-neostigmine treated contractions by ILJ (25 mg/ml) were 15.16 9 1.14 and 17.00 9 1.15 mm (n=6), respectively. In cold (4°C for 96 h)-induced denervated tissues (Fig. 7), ILJ (25 mg/ml)-induced contraction of guinea pig ileum was significantly augmented from 15.59 1.23 (fresh) to 54.5093.51 mm, but
Table 1 ED50 values and doses to produce Emax of acetylcholine, carbachol, isoprenaline and Ipomoea carnea leaf juice(ILJ) in guinea pig ileum(mean with 95% C.L. in parenthesis, n= 6) Response
Drug
ED50 (ng/ml)
Emax dose (ng/ml)
Contraction
Acetylcholine Carbachol ILJ
14.08 (8.76–19.39) 13.64 (9.81–17.47) 26.38×104 ((11.70–41.05)×104)
23.3×102 ((12.5–34.2)×102) 10.0×102 10.0×106
Relaxation
Isoprenaline ILJ
7.00 (5.92–8.08) 11.69×104 ((10.23–13.16)×104)
2.7×102 ((1.8–3.5)×102) 7.67×106 ((6.19–9.15)×106)
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ACh (2 ng/ml)-induced response remained unaltered (24.1791.49 and 27.6792.01 mm in fresh and cooled tissues, respectively, n =6). In these tissues, ILJ (25 mg/ml) could not produce relaxation, but induced sustained contraction which was suppressed by atropine (1 mg/ml) by 75.6% (from 54.5093.51 to 13.3391.20 mm, n = 6).
3.2. Relaxatory phase of ILJ Atropine (1 mg/ml, 15 min) abolished the relaxatory phase of ILJ (25 mg/ml) in 12.5% tissues,
Fig. 7. Acetylcholine (A, 2 ng/ml)- and I. carnea leaf juice (I, 25 mg/ml)-induced contractions of fresh (f) and cold-induced denervated (c) guinea pig ileal preparations and effect of atropine (T, 1 mg/ml) on I. carnea leaf juice (I, 25 mg/ml)-induced contraction of latter tissues (n = 6). *PB0.05 and cPB 0.05 in comparison to If and Ic, respectively.
Fig. 5. Acetylcholine (A, 2 ng/ml)-induced contractions in absence and presence of lignocaine (L, 50 mg/ml) and neostigmine (N, 30 ng/ml) in guinea pig ileum (n= 6). *PB 0.05.
Fig. 8. I. carnea leaf juice (I, 25 mg/ml)-induced relaxations in absence and presence of atropine (T, 1 mg/ml), phenoxynezamine (B, 35 mg/ml), propranolol (P, 1 mg/ml) or lignocaine (L, 50 mg/ml) and adrenaline (D, 0.3 mg/ml)-induced relaxations in absence and presence of lignocaine (L, 50 mg/ml) in guinea pig ileum (n =6). *P B0.05 in comparison to respective controls.
Fig. 6. Individual and combined effects of I. carnea leaf juice (I, 25 mg/ml), acetylcholine (A, 2 ng/ml) and carbachol (C, 2 ng/ml) on guinea pig ileum (n= 6).
whereas the response was not blocked in rest (87.5%) of the preparations (Fig. 1, n= 16). In presence of atropine in latter tissues, the ILJ (25 mg/ml)-induced relaxation was not significantly altered by phenoxybenzamine (35 mg/ml, 15 min; from 15.8391.17 to 18.169 1.53 mm, n= 6), but was reduced from 18.3391.47 to 9.179 0.98 mm by propranolol (1 mg/ml, 10 min, n= 6; Fig. 8). Propranolol (1 mg/ml, 10 min), in absence of
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atropine, significantly increased the ILJ (25 mg/ ml)-induced contraction from 16.339 0.76 to 53.6793.82 mm without producing any relaxation of guinea pig ileum (n= 6; Fig. 4). Lignocaine (50 mg/ml, 5 min) abolished ILJ (25 mg/ml)-induced but not adrenaline (0.3 mg/ml; from 15.3391.45 to 14.6691.71 mm, n = 6)-induced relaxation (Fig. 8).
3.3. Final contractile phase of ILJ Atropine could not affect the third phase (slow and sustained contraction) of ILJ-mediated response in guinea pig ileum (Fig. 1). The responses of ILJ (25 mg/ml) in absence and presence of atropine (1 mg/ml) were 31.1792.44 and 30.179 2.41 mm, respectively (n = 6).
4. Discussion
4.1. Initial contractile phase of ILJ It is evident from the results that ILJ contracted the guinea pig ileum dose-dependently and its initial contractile phase was blocked by a muscarinic receptor antagonist, atropine. This finding indicates the involvement of cholinergic muscarinic receptors in producing initial contraction by ILJ in guinea pig ileum. There seems to be one or more than one of the three possible mechanisms responsible for this contractile action of ILJ — (i) stimulation of ACh release from the cholinergic nerve endings; (ii) inhibition of acetylcholinesterase (AChE) enzyme at the neuro-effector junction; or (iii) direct activation of the muscarinic receptors of guinea pig ileal smooth muscle. Lignocaine, a membrane stabilizer, blocks the axonal conduction and thereby neurotransmitter release at a concentration of 50 mg/ml (Mishra and Raviprakash, 1980). In our study, the same dose of lignocaine could not affect the contraction produced by ILJ or ACh. Therefore, it is an indirect evidence that there was possibly no stimulation of ACh release at the prejunctional site by ILJ or ACh. This view is further strengthened by the finding that the contractile responses of ILJ
and ACh were not reduced in cooled preparations. The method of cooling provides a means of obtaining a smooth nuscle preparation free from the neuronal influences (Ambache, 1946). Hence, the ILJ mediated initial contraction was a nonneurogenic response. Neostigmine, an anticholinesterase agent (AntiChE), though significantly increased the ACh-induced contraction, failed to alter the degree of the contractile response of ILJ. The combination of ACh and an Anti-ChE produces a potentiated effect (Kela et al., 1995) because the percent of ACh molecules, which is normally destroyed by AChE enzyme in absence of Anti-ChE, becomes available to act on the receptors to produce extra effect. In the present study, ACh and ILJ produced additive effect when they were used together. The above observations nullify the probable involvement of AChE enzyme in the ILJ-induced initial contraction. Therefore, there is a maximum possibility that ILJ directly activated the muscarinic receptors of guinea pig ileum preparation. Carbachol, a choline ester, binds with the muscarinic receptor for its agonistic action in smooth muscle, whereas atropine competes with ACh and carbachol for a common binding site on the muscarinic receptor (Brown and Taylor, 1996). In this study, carbachol produced additive effects with ACh or ILJ. The individual responses of carbachol, ACh and ILJ were blocked by atropine. It seems from these findings that ILJ binds with the same site of the receptor as for ACh, carbachol and atropine in guinea pig ileum. A similar finding of cholinergic involvement of I. carnea-induced contraction of frog rectus abdominis and bradycardia of rabbit heart was reported by Abdelhadi et al., 1989).
4.2. Relaxatory phase of ILJ ILJ produced dose-dependent relaxation at the second phase of its response. In most of the tissues, atropine did not modulate the ILJ-induced relaxation. In atropine-primed preparations, ILJ-mediated relaxation was not altered by phenoxybenzamine, a non-specific a-adrenoceptor blocker, but reduced by a b-adrenoceptor antagonist, propranolol. On the other hand, propranolol
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alone significantly increased the ILJ-induced initial contraction without producing any relaxation. Hence the relaxatory phase of ILJ may be due to activation of b-adrenoceptors in guinea pig ileum. Lignocaine (50 mg/ml) abolished the ILJ-induced, but not adrenaline-induced relaxation of guinea pig ileum. Therefore, this relaxatory phase of ILJ is appeared to be a neurogenic response. This hypothesis is further supported by our finding that the relaxatory response of ILJ was abolished in cooled preparations. From the above observations, the probability of the stimulation of adrenergic nerve endings by ILJ to release noradrenaline can not be ruled out. However, surprisingly, in 12.5% tissues, the relaxatory effect of ILJ was blocked by atropine. We are unable to explain the phenomenon of this observation.
4.3. Final contractile phase of ILJ The final contractile phase of ILJ in guinea pig ileum remained unaltered in presence of atropine, indicating that the effect was possibly mediated by a non-cholinergic mechanism. Thus it may be concluded from the results of the present investigation that ILJ-induced initial contraction was due to modulation of the cholinergic system of guinea pig ileum and this phenomenon probably corelates with the hypermotility of gut by I. carnea leaves. Possible involvement of adrenergic and non-cholinergic mechanisms in relaxatory and final contractile phases of ILJ, respectively are also suggested. Fractionation of the active principles of ILJ and elucidation of their individual mechanism of actions merit further investigation.
Acknowledgements Authors are thankful to the Dean, College of Veterinary Science and A.H., Anjora, Durg, for providing necessary facilities to conduct the study. .
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Thanks are also due to Ram Gopal Sharma for laboratory assistance.
References Abdelhadi, A.A., el-Kheir, Y.M., Hassan, T., 1989. A succinylcholine-like action of an Ipomoea carnea Jacq. sub sp. fistulosa (Mart. ex choisy) extract. Pharmacological Research 21, 431 – 437. Adam, S.E.I., Tartour, G., Obied, H.M., Idris, O.F., 1973. Effect of Ipomoea carnea on liver and serum enzymes in young ruminants. Journal of Comparative Pathology 83, 531 – 542. Ambache, N. 1946. Journal of Physiology. 104, 266 – 287. Cited in Mishra and Raviprakash (1980). Brown, J.H., Taylor, P., 1996. Muscarinic receptor agonists and antagonists. In: Hardman, J.G., Limbird, L.E., Molinoff, P.B., Ruddon, R.W., Gilman, A.G. (Eds.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 9th ed. McGraw-Hill Publication, New York, pp. 141 – 160. Damir, H.A., Adam, S.E.I., Tartour, G., 1987. The effects of Ipomoea carnea on goats and sheep. Veterinary and Human Toxicology 29, 316 – 319. Gahlot, A.K., Gupta, H.K., 1984. Toxicity of the plant ‘Vilayati akadi’ (Ipomoea carnea) in livestock. Indian Journal of Veterinary Medicine 4, 142. Ghosh, M.N., 1984. Fundamentals of Experimental Pharmacology. Scientific Book Agency, Calcutta. Kela, A.K., Sharma, A.K., Mehta, V.L., 1995. Does ethanol release acetylcholine to produce contraction on isolated frog rectus abdominis. Indian Journal of Pharmacology 27, 195 – 196. Lum, B.K.B., Kermani, M.H., Heilman, R.D., 1966. Intestinal relaxation produced by sympathomimetic amines in the isolated rabbit jejunum: selective inhibition by adrenergic blocking agents and by cold storage. Journal of Pharmacology and Experimental Therapeutics 154, 463 – 471. Mishra, S.K., Raviprakash, V., 1980. Possible involvement of a cholinergic mechanism in calcium-induced contractions of chick oesophagus. Journal of Pharmacy Pharmacology 32, 716 – 717. Shrivastava, D.N., Soni, J.L., 1987. Clinical Veterinary Toxicology and Jurisprudence. Anubha Prakashan, Jabalpur, India. Snedecor, G.W., Cochran, W.G. 1967. Statistical Methods, sixth ed., Oxford and IBH, New Delhi. Srilatha, C., Naidu, N.R.G., Rao, P.R., 1993. Symptomatology of Ipomoea carnea plant toxicity in goats. Journal of Veterinary and Animal Sciences 24, 201 – 202. Tirkey, K., Yadava, K.P., Jha, G.J., Barerjee, N.C., 1987. Effect of feeding of Ipomoea carnealpomoea leaves in goats. Indian Journal of Animal Science 57, 863 – 866.
Possible involvement of cholinergic and adrenergic mechanisms in changing contractility of guinea pig ileum by Ipomoea carnea S.K. Hore *, Ram Ottalwar, K.M. Koley, A.K. Pathak Department of Pharmacology and Toxicology, College of Veterinary Science and A.H., I.G.A.U., Anjora, Durg-491001 (MP), India Received 12 February 1999; received in revised form 4 January 2000; accepted 14 January 2000
Abstract The study aimed to elucidate the mechanism (s) of action of Ipomoea carnea leaf juice (ILJ) in changing contractility of guinea pig ileum. ILJ produced dose-dependent (10 – 10 000 mg/ml) triphasic responses. The initial contractile phase was blocked by atropine (1 mg/ml) but had additive effect with acetylcholine (2 ng/ml) or carbachol (2 ng/ml). Neostigmine (30 ng/ml) or lignocaine (50 mg/ml) failed to alter the response. In cold-induced denervated preparations, this phase was augmented. The relaxatory phase of ILJ was not modified by phenoxybenzamine (35 mg/ml) but was reduced by propranolol (1 mg/ml) and abolished by lignocaine (50 mg/ml). The final contractile phase of ILJ was not affected by atropine (1 mg/ml). These results suggest that the triphasic response of ILJ is possibly mediated through cholinergic, adrenergic and non-cholinergic mechanisms, respectively. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ipomoea carnea leaf juice; Guinea pig ileum; Triphasic response; Cholinergic; Adrenergic; Non-cholinergic
1. Introduction The plant Ipomoea carnea, Jacq. syn. Argyreia superbiens, Cave (Convolvulaceae), besharam in hindi, is found throughout India. Normally, animals refuse to take the plant, because it is not a palatable forage for them (Gahlot and Gupta, * Corresponding author. Present address: Department of Pharmacology and Toxicology, College of Veterinary Science, G.B. Pant University of Agriculture and Technology, Pantnagar-263 145 (U.P.), India.
1984). But in scarcity, like draught condition, they ingest the plant and thereby get poisoned (Shrivastava and Soni, 1987). Both fresh and dry leaves and stems of the plant are equally toxic to animals (Damir et al., 1987). Goats and sheep followed by cattle are the most common livestock species affected (Adam et al., 1973; Gahlot and Gupta, 1984). Beside other signs, the digestive disorders exhibited in the form of ruminal hypermotility and diarrhoea are the common symptoms of poisoning (Gahlot and Gupta, 1984; Tirkey et al., 1987;
0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 1 7 3 - 2
254
S.K. Hore et al. / Journal of Ethnopharmacology 71 (2000) 253–259
Srilatha et al., 1993). The mechanism (s) involved in altering the gastrointestinal tract contractility by I. carnea is not yet clear. The present study has, therefore, aimed to investigate the mechanism (s) of action of I. carnea leaf juice (ILJ) to produce hypermotility of gut.
32°C and continuously bubbled with air. The preparations were allowed to equilibrate under a resting tension of 0.5 g for 60 min, during which the bathing solution was changed every 15 min. The isotonic contractions (magnification × 8) were recorded on a slow moving (0.025 mm/s) smoked Kymograph paper using a frontal writing lever.
2. Materials and methods
2.5. Elucidation of mechanism(s) of action of ILJ 2.1. Plant material Mature green leaves, collected in the month of March–April from the College campus, were identified and authenticated by Dr D. Karkun, Head of Botany Department, College of Science, Arts and Commerce, Durg, MP, India.
2.2. Preparation of juice One hundred grams of fresh leaves was triturated with 100 ml of distilled water and the juice was centrifuged at 3000 rpm for 15 min. The supernatant was referred to as I. carnea leaf juice (ILJ) and was used for this experiment. The w/w yield of prepared extract was 12.3% in terms of starting crude material. The concentration of extract in the supernatant was 10.4 mg/ml.
2.3. Animals Adult guinea pigs of either sex (300 – 350 g) obtained from the Laboratory Animal Unit of the Department were used in the study. They were maintained under controlled conditions with a standard pellet diet and water ad libitum.
2.4. Preparation of guinea pig ileum The animals were sacrificed by a sharp blow on the head and cutting the neck blood vessels (Ghosh, 1984). The terminal segments (2 cm long) of ileum after discarding 10 cm from the ileocaecal junction, were immediately removed and cleaned. The tissues were then mounted in a 20 ml organ bath containing Ringer – Locke solution (composition in mM: NaCl 154, KCl 5.6, CaCl2 2.2, NaHCO3 6.0 and glucose 11.1) maintained at
To elucidate the mechanism(s) of action of ILJ in guinea pig ileum the steps given below were followed. Each tissue preparation was exposed to only one agonist or one combination. 1. Dose-dependent contractions induced by ILJ (10–10 000 mg/ml), acetylcholine (0.3–1000 ng/ml) and carbachol (1–1000 ng/ml) were recorded. 2. Atropine sulphate (1 mg/ml) was added to the bath fluid 15 min before recording the response of ILJ (25 mg/ml), acetylcholine (ACh, 2 ng/ml) or carbachol (2 ng/ml) to antagonize their effects. 3. The response of ILJ (25 mg/ml), ACh (2 ng/ml) and adrenaline (0.3 mg/ml) were taken before and after incubating the tissue with lignocaine (50 mg/ml) for 5 min. 4. The combined effect of ILJ (25mg/ml) and ACh (2 ng/ml) were observed. 5. The ACh (2 ng/ml) and ILJ (25 mg/ml)-induced contractions were recorded in absence and presence of neostigmine (30 ng/ml, 15 min). 6. The effects of carbachol (2 ng/ml) were seen either alone or in combination with ILJ (25 mg/ml) or ACh (2 ng/ml). 7. Effect of isoprenaline on acetylcholine (1 mg/ ml)-induced contractions was recorded. 8. The ILJ (25 mg/ml) and adrenaline (0.3mg/ml)mediated responses were observed before and after incubating the preparations with either phenoxybenzamine (35 mg/ml, 15 min) or propranolol (1 mg/ml, 10 min) in absence or presence of atropine (1 mg/ml). 9. Several experiments were conducted on the preparations that had been stored at low temperature of 4°C for 96 h for denervation (Lum
S.K. Hore et al. / Journal of Ethnopharmacology 71 (2000) 253–259
255
et al., 1966), but were later maintained at 32°C bath temperature during experiment.
2.6. Drugs Acetylcholine chloride (Sigma), L-adrenaline (Loba Chemie), atropine sulphate (Loba Chemie), carbachol (Sigma), isoprenaline hydrochloride (Sigma), lignocaine hydrochloride (Astra-IDL), neostigmine methylsulphate (Tablets India), Phenoxybenzamine hydrochloride (SKF), and propranolol hydrochloride (I.C.I.) were used in the experiment.
2.7. Statistical analysis The experimental data were analysed by the Student’s t-test for obtaining the level of significance. PB 0.05 was considered as significant. ED50 values were determined by regression analysis, using the least square methods and were expressed as mean with 95% confidence limits (Snedecor and Cochran, 1967).
3. Results I. carnea leaf juice (ILJ, 25 – 100 mg/ml) produced either triphasic (83.3%) or biphasic (16.7%) responses in guinea pig ileum. In the former group of tissues, the triphasic response consisted
Fig. 2. Dose-contraction curves of (a) acetylcholine (ACh) and carbachol (CB); and (b) I. carnea leaf juice (ILJ) in respect to percent maximum response of ACh in guinea pig ileum (n = 6).
of a fast contraction followed by a brief relaxation and then a sustained contcentration (Fig. 1). A dose-dependent contraction followed by relaxation was produced by ILJ. The dose-response curves (n= 6) of contractile effects of ACh, carbachol and ILJ and relaxatory effect of ILJ have been presented in Figs. 2 and 3. The ED50 values and the contractions required to produce maximum response (Emax) of the above drugs have been listed in Table 1. Fig. 1. (a) Triphasic response of I. carnea leaf juice (, 25 mg/ml) on guinea pig ileum. (b) Blockade of initial contractile phase of I. carnea leaf juice (, 25 mg/ml) by atropine ( , 1 mg/ml). W, wash.
3.1. Initial contractile phase of ILJ Carbachol (2 ng/ml), ACh (2 ng/ml) and ILJ (25 mg/ml) contracted the guinea pig ileum by
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Fig. 4. I. carnea leaf juice (I, 25 mg/ml)-induced contractions in absence and presence of lignocaine (L, 50 mg/ml), neostigmine (N, 30 ng/ml) and propranolol (P, 1 mg/ml) in guinea pig ileum (n =6). *PB 0.05.
Fig. 3. Dose-relaxation curves of (a) isoprenaline (ISP) to acetylcholine (ACh, 1 mg/ml)-induced contraction; and (b) I. carnea leaf juice (ILJ) in guinea pig ileum (n= 6).
29.59 2.96, 24.17 9 1.49 and 15.3 9 1.63 mm, respectively (Fig. 6), which were about 16.7, 13.8 and 9.1% of their respective maximum responses. These effects were completely blocked by atropine (1 mg/ml). The combination of carbachol (2 ng/ ml) and ACh (2 ng/ml), carbachol (2 ng/ml) and ILJ (25 mg/ml), or ACh (2 ng/ml) and ILJ (25 mg/ml) produced additive responses (52.33 93.61,
44.179 2.14 or 40.509 1.18 mm, respectively, Fig. 6, n= 6). Lignocaine (50 mg/ml) failed to block the contractile responses of both ILJ (25 mg/ml, Fig. 4) and ACh (2 ng/ml, Fig. 5), but significantly blocked ILJ (25 mg/ml)-induced relaxation phase (Fig. 8). Though neostigmine (30 ng/ml, 15 min) significantly increased the ACh (2 ng/ml)-induced contraction from 26.169 1.05 to 70.8393.47 mm (n= 6, Fig. 5), but failed to alter the response of ILJ (25 mg/ml, Fig. 4). The preand post-neostigmine treated contractions by ILJ (25 mg/ml) were 15.16 9 1.14 and 17.00 9 1.15 mm (n=6), respectively. In cold (4°C for 96 h)-induced denervated tissues (Fig. 7), ILJ (25 mg/ml)-induced contraction of guinea pig ileum was significantly augmented from 15.59 1.23 (fresh) to 54.5093.51 mm, but
Table 1 ED50 values and doses to produce Emax of acetylcholine, carbachol, isoprenaline and Ipomoea carnea leaf juice(ILJ) in guinea pig ileum(mean with 95% C.L. in parenthesis, n= 6) Response
Drug
ED50 (ng/ml)
Emax dose (ng/ml)
Contraction
Acetylcholine Carbachol ILJ
14.08 (8.76–19.39) 13.64 (9.81–17.47) 26.38×104 ((11.70–41.05)×104)
23.3×102 ((12.5–34.2)×102) 10.0×102 10.0×106
Relaxation
Isoprenaline ILJ
7.00 (5.92–8.08) 11.69×104 ((10.23–13.16)×104)
2.7×102 ((1.8–3.5)×102) 7.67×106 ((6.19–9.15)×106)
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ACh (2 ng/ml)-induced response remained unaltered (24.1791.49 and 27.6792.01 mm in fresh and cooled tissues, respectively, n =6). In these tissues, ILJ (25 mg/ml) could not produce relaxation, but induced sustained contraction which was suppressed by atropine (1 mg/ml) by 75.6% (from 54.5093.51 to 13.3391.20 mm, n = 6).
3.2. Relaxatory phase of ILJ Atropine (1 mg/ml, 15 min) abolished the relaxatory phase of ILJ (25 mg/ml) in 12.5% tissues,
Fig. 7. Acetylcholine (A, 2 ng/ml)- and I. carnea leaf juice (I, 25 mg/ml)-induced contractions of fresh (f) and cold-induced denervated (c) guinea pig ileal preparations and effect of atropine (T, 1 mg/ml) on I. carnea leaf juice (I, 25 mg/ml)-induced contraction of latter tissues (n = 6). *PB0.05 and cPB 0.05 in comparison to If and Ic, respectively.
Fig. 5. Acetylcholine (A, 2 ng/ml)-induced contractions in absence and presence of lignocaine (L, 50 mg/ml) and neostigmine (N, 30 ng/ml) in guinea pig ileum (n= 6). *PB 0.05.
Fig. 8. I. carnea leaf juice (I, 25 mg/ml)-induced relaxations in absence and presence of atropine (T, 1 mg/ml), phenoxynezamine (B, 35 mg/ml), propranolol (P, 1 mg/ml) or lignocaine (L, 50 mg/ml) and adrenaline (D, 0.3 mg/ml)-induced relaxations in absence and presence of lignocaine (L, 50 mg/ml) in guinea pig ileum (n =6). *P B0.05 in comparison to respective controls.
Fig. 6. Individual and combined effects of I. carnea leaf juice (I, 25 mg/ml), acetylcholine (A, 2 ng/ml) and carbachol (C, 2 ng/ml) on guinea pig ileum (n= 6).
whereas the response was not blocked in rest (87.5%) of the preparations (Fig. 1, n= 16). In presence of atropine in latter tissues, the ILJ (25 mg/ml)-induced relaxation was not significantly altered by phenoxybenzamine (35 mg/ml, 15 min; from 15.8391.17 to 18.169 1.53 mm, n= 6), but was reduced from 18.3391.47 to 9.179 0.98 mm by propranolol (1 mg/ml, 10 min, n= 6; Fig. 8). Propranolol (1 mg/ml, 10 min), in absence of
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atropine, significantly increased the ILJ (25 mg/ ml)-induced contraction from 16.339 0.76 to 53.6793.82 mm without producing any relaxation of guinea pig ileum (n= 6; Fig. 4). Lignocaine (50 mg/ml, 5 min) abolished ILJ (25 mg/ml)-induced but not adrenaline (0.3 mg/ml; from 15.3391.45 to 14.6691.71 mm, n = 6)-induced relaxation (Fig. 8).
3.3. Final contractile phase of ILJ Atropine could not affect the third phase (slow and sustained contraction) of ILJ-mediated response in guinea pig ileum (Fig. 1). The responses of ILJ (25 mg/ml) in absence and presence of atropine (1 mg/ml) were 31.1792.44 and 30.179 2.41 mm, respectively (n = 6).
4. Discussion
4.1. Initial contractile phase of ILJ It is evident from the results that ILJ contracted the guinea pig ileum dose-dependently and its initial contractile phase was blocked by a muscarinic receptor antagonist, atropine. This finding indicates the involvement of cholinergic muscarinic receptors in producing initial contraction by ILJ in guinea pig ileum. There seems to be one or more than one of the three possible mechanisms responsible for this contractile action of ILJ — (i) stimulation of ACh release from the cholinergic nerve endings; (ii) inhibition of acetylcholinesterase (AChE) enzyme at the neuro-effector junction; or (iii) direct activation of the muscarinic receptors of guinea pig ileal smooth muscle. Lignocaine, a membrane stabilizer, blocks the axonal conduction and thereby neurotransmitter release at a concentration of 50 mg/ml (Mishra and Raviprakash, 1980). In our study, the same dose of lignocaine could not affect the contraction produced by ILJ or ACh. Therefore, it is an indirect evidence that there was possibly no stimulation of ACh release at the prejunctional site by ILJ or ACh. This view is further strengthened by the finding that the contractile responses of ILJ
and ACh were not reduced in cooled preparations. The method of cooling provides a means of obtaining a smooth nuscle preparation free from the neuronal influences (Ambache, 1946). Hence, the ILJ mediated initial contraction was a nonneurogenic response. Neostigmine, an anticholinesterase agent (AntiChE), though significantly increased the ACh-induced contraction, failed to alter the degree of the contractile response of ILJ. The combination of ACh and an Anti-ChE produces a potentiated effect (Kela et al., 1995) because the percent of ACh molecules, which is normally destroyed by AChE enzyme in absence of Anti-ChE, becomes available to act on the receptors to produce extra effect. In the present study, ACh and ILJ produced additive effect when they were used together. The above observations nullify the probable involvement of AChE enzyme in the ILJ-induced initial contraction. Therefore, there is a maximum possibility that ILJ directly activated the muscarinic receptors of guinea pig ileum preparation. Carbachol, a choline ester, binds with the muscarinic receptor for its agonistic action in smooth muscle, whereas atropine competes with ACh and carbachol for a common binding site on the muscarinic receptor (Brown and Taylor, 1996). In this study, carbachol produced additive effects with ACh or ILJ. The individual responses of carbachol, ACh and ILJ were blocked by atropine. It seems from these findings that ILJ binds with the same site of the receptor as for ACh, carbachol and atropine in guinea pig ileum. A similar finding of cholinergic involvement of I. carnea-induced contraction of frog rectus abdominis and bradycardia of rabbit heart was reported by Abdelhadi et al., 1989).
4.2. Relaxatory phase of ILJ ILJ produced dose-dependent relaxation at the second phase of its response. In most of the tissues, atropine did not modulate the ILJ-induced relaxation. In atropine-primed preparations, ILJ-mediated relaxation was not altered by phenoxybenzamine, a non-specific a-adrenoceptor blocker, but reduced by a b-adrenoceptor antagonist, propranolol. On the other hand, propranolol
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alone significantly increased the ILJ-induced initial contraction without producing any relaxation. Hence the relaxatory phase of ILJ may be due to activation of b-adrenoceptors in guinea pig ileum. Lignocaine (50 mg/ml) abolished the ILJ-induced, but not adrenaline-induced relaxation of guinea pig ileum. Therefore, this relaxatory phase of ILJ is appeared to be a neurogenic response. This hypothesis is further supported by our finding that the relaxatory response of ILJ was abolished in cooled preparations. From the above observations, the probability of the stimulation of adrenergic nerve endings by ILJ to release noradrenaline can not be ruled out. However, surprisingly, in 12.5% tissues, the relaxatory effect of ILJ was blocked by atropine. We are unable to explain the phenomenon of this observation.
4.3. Final contractile phase of ILJ The final contractile phase of ILJ in guinea pig ileum remained unaltered in presence of atropine, indicating that the effect was possibly mediated by a non-cholinergic mechanism. Thus it may be concluded from the results of the present investigation that ILJ-induced initial contraction was due to modulation of the cholinergic system of guinea pig ileum and this phenomenon probably corelates with the hypermotility of gut by I. carnea leaves. Possible involvement of adrenergic and non-cholinergic mechanisms in relaxatory and final contractile phases of ILJ, respectively are also suggested. Fractionation of the active principles of ILJ and elucidation of their individual mechanism of actions merit further investigation.
Acknowledgements Authors are thankful to the Dean, College of Veterinary Science and A.H., Anjora, Durg, for providing necessary facilities to conduct the study. .
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Thanks are also due to Ram Gopal Sharma for laboratory assistance.
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