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Anti-inflammatory activity of Crinum asiaticum plant and its effect on bradykinin-induced contractions on isolated uterus
Immunopharmacology 43 Ž1999. 311–316 www.elsevier.comrlocaterimmpharm
Anti-inflammatory activity of Crinum asiaticum plant and its effect on bradykinin-induced contractions on isolated uterus Awatef M. Samud, M. Zaini Asmawi, Jagdish N. Sharma ) , Ahmad Pauzi M. Yusof Department of Pharmacology and Physiology, School of Pharmaceutical Sciences, UniÕersiti Sains Malaysia, 11800 Penang, Malaysia Accepted 3 June 1999
Abstract Crinum asiaticum Linn plant is used in Malaysia as a rheumatic remedy and to relieve local pain. In the present study, we examined the anti-inflammatory effects of this plant extract on carrageenan-induced hind paw oedema in mice. C. asiaticum was serially extracted with petroleum ether, followed by chloroform and lastly, methanol. The chloroform and methanol extracts of the plant given orally Ž50 mg kgy1 . caused significant Ž p - 0.05; n s 7. reduction in paw oedema but the petroleum ether extract did not induce significant effect Ž p ) 0.05. on paw oedema. The methanol extract was then dissolved in water and extracted consecutively with chloroform, ethyl acetate and butanol. The chloroform fraction of methanol extract ŽCFME. treatment Ž50 mg kgy1 . significantly reduced Ž p - 0.05; n s 7. the acute paw oedema. This may indicate that active anti-inflammatory compounds are present in the CFME. In an attempt to study the mechanism of action of its anti-inflammatory activity, the effects of CFME on BK- and histamine-induced contractions were investigated in isolated rat uterus and guinea-pig ileum preparations, respectively. It was found that CFME caused dose-dependent reduction Ž p - 0.05; n s 6. of the contractile response induced by BK and shifted the log dose–response curve of histamine to the right. The present findings suggest that C. asiaticum possessed an anti-inflammatory activity as suggested by its use in traditional medicine. The anti-inflammatory activity of this plant could not have been due to its anti-bradykinin activities as CFME non-specifically inhibited BK-induced contraction. It also suggest that CFME may contain compoundŽs. with anti-histaminic properties. The significance of these findings is discussed. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Anti-inflammatory activity; Carrageenan oedema; C. asiaticum; Bradykinin responses; Rat uterus
1. Introduction Crinum asiaticum Linn Žfamily: Amaryllidaceae. is a herbaceous plant of small to moderate size with greenish-feathery leaves. In Southeast Asian counAbbreÕiations: BK, Bradykinin; CMC, Carboxymethycellulose; CFME, Chloroform fraction of methanol extract of C. asiaticum plant ) Corresponding author. Fax: q60-604-6570017; e-mail: [email protected]
tries, C. asiaticum has a considerable medicinal reputation as a potent folk medicine in the treatment of injury and inflamed joints ŽBurkill, 1966.. In the Philippines, the leaves are prepared as emollient for external use to treat inflamed joints and sprains ŽValenzuela et al., 1930.. In early times, it is used in Indonesia and in Malay peninsula as an antidote for wounds from poisoned arrows. The poultice Žbroader leaves, oiled and heated. are applied to swollen joints and used to treat fever, headache and local pain ŽBurkill, 1966..
0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 9 . 0 0 1 3 2 - 0
312
A.M. Samud et al.r Immunopharmacology 43 (1999) 311–316
Although few pharmacological studies have been reported ŽOchi et al., 1976; Ghosal et al., 1985., this plant has not been subjected to systematic pharmacological anti-inflammatory evaluation to support its use. The present study, therefore, was carried out to evaluate the effect of C. asiaticum plant on an acute inflammatory model using carrageenan-induced oedema in mice. The BK and histamine are important inflammatory mediators. These mediators possess potent smooth muscle contracting properties. Hence, the effects of the plant extract on the contractile responses induced by BK and histamine on
isolated smooth muscle preparations were examined in an attempt to elucidate the involvement of these two mediators in the anti-inflammatory activity.
2. Materials and methods 2.1. Identification of plant material and preparation of extract Samples of fresh green leaves of C. asiaticum plant were collected in November, 1996 from Penang
Fig. 1. Fractionation of crude methanol extract of C. asiaticum.
A.M. Samud et al.r Immunopharmacology 43 (1999) 311–316
island, Malaysia. This plant had been identified and voucher specimens was deposited in the herbarium of the School of Biological Sciences, Universiti Sains Malaysia Žspecimen No. 10106.. C. asiaticum dried powdered leaves Ž950 g. were refluxed in Soxhlet’s apparatus with petroleum ether at 40–608C, chloroform at 45–558C and finally with methanol at 60–708C. The mark left after each extraction was completely dried from the previous solvent of extraction. The extracts were filtered and the filtrate were evaporated under reduced pressure ŽRotavapor, Buchi. The residues obtained were dried under vacuum at y1108C ŽVacuum system B-169, Buchi. for 10 h. The yield of the extraction procedure were 40.85 g Ž4.31%. for petroleum ether extract, 26.89 g Ž2.83%. for chloroform extract and 146.97 g Ž15.47%. for methanol extract. Using the maceration technique ŽTharib et al., 1983., the methanol extract was further fractionated according to the scheme shown in Fig. 1. The chloroform fraction which was obtained from the methanol extract is referred to hereafter as CFME.
313
2.3. Isolated preparations 2.3.1. Rat uterus Virgin rats Ž120–200 g; n s 6. were brought to artificial oestrus 18–22 h after a single S.C. injection of stilboestrol ŽSigma. 0.1 mg kgy1 solution in ethanol. Rat were killed and horns of uterus were suspended in De Jalon’s solution at 358C as described by Sharma and Zeitlin Ž1977.. BK doses-contractile responses were obtained in the presence and absence of CFME of C. asiaticum plant. CFME was tested at concentration of 1, 5, 10 and 20 mg mly1 . BK ŽSigma. was used at doses of 1, 2, 4 and 8 pg mly1 . 2.3.2. Guinea-pig ileum preparation Guinea-pig Ž250–350 gm; n s 6. were killed by a blow on the head and cutting the throat. The strip of ileum 2–3 cm was suspended in Tyrode’s solution at 378C ŽFloyd, 1971.. A dose contractile responses of histamine was obtained in the absence and presence of CFME of C. asiaticum plant. Histamine was used at doses of 0.1, 0.2, 0.4, 0.8 and 1.6 mg mly1 .
2.2. Carrageenan-induced oedema The procedure used to assess anti-inflammatory activity was based on the method used by Winter et al. Ž1962.. Food was withdrawn from the animals 24 h prior to the experiments, but were supplied with water ad libitum. Plant extract 50 mg kgy1 , indomethacin ŽSigma. 1 mg kgy1 suspended in 1% CMC as positive control and vehicle as negative control were administered orally to groups of mice Ž20–35 g; n s 7 each.. One hour later, oedema was induced by injecting in the right hind paw 0.02 ml carrageenan ŽSigma. 1% Ž100 mg carrageenan dissolved in 10 ml saline.. The pad thickness of hind paw was measured by micrometer Žmm; Mitutoyo. before and 3 h after S.C. injection of carrageenan. The anti-inflammatory activity of the plant extract and indomethacin to inhibit the swelling of the hind paw was calculated by the following formula: % inflammations Ž A y B . rB = 100 where A s measurement of pad thickness 3 h after carrageenan-induced oedema, B s initial measurement of pad thickness before carrageenan-induced oedema.
Fig. 2. Effect of oral administration of indomethacin Ž1 mg kgy1 ., petroleum ether extract Ž2 g kgy1 ., chloroform extract Ž50 mg kgy1 . and methanol extract Ž50 mg kgy1 . of plant C. asiaticum after 3 h of carrageenan-induced oedema in mice hind paw. Results are expressed as mean percentage of inflammation" S.E.M. U Significant difference Ž p- 0.05. from the control. Seven mice were used in each group.
314
A.M. Samud et al.r Immunopharmacology 43 (1999) 311–316
2.3.3. Statistical analysis Data were expressed as the mean " S.E.M. The data were evaluated for significance of difference using analysis of variance ŽANOVA.. The homogeneity of groups was verified by Duncan’s test at an alpha level equal to 5%.
3. Results
3.1. Effects of plant extracts on paw oedema
Fig. 3. Effect of oral administration of indomethacin Ž1 mg kgy1 .; n-butanol fraction Ž50 mg kgy1 ., chloroform fraction Ž50 mg kgy1 ., ethyl acetate fraction Ž50 mg kgy1 ., and water fraction Ž50 mg kgy1 . obtained from methanol extract of plant C. asiaticum after 3 h of carrageenan-induced oedema in mice hind paw. Results are expressed as mean percentage of inflammation" S.E.M. U Significant difference Ž p- 0.05. from the control. Seven mice were used in each group.
CFME was tested at doses of 5, 10, 20 and 50 mg mly1 .
Results in Fig. 2 shows that methanol and chloroform extracts Ž50 mg kgy1 . and indomethacin Ž1 mg kgy1 . caused significant Ž p - 0.05. inhibition of paw oedema Ž94.8%, 86.7% and 72.0%, respectively. as compared to the non-treated control group of mice. Petroleum ether extract, 2 gm kgy1 did not significantly Ž p ) 0.05. inhibit carrageenan-induced inflammation. Fig. 3 shows that CFME 50 mg kgy1 significantly inhibited carrageenan-induced oedema Ž p - 0.05.. The inhibitory potency was about the same as that of indomethacin 1 mg kgy1 . However, butanol, ethyl acetate and aqueous fractions Ž50 mg kgy1 . of the methanol extract did not cause signifi-
Fig. 4. Dose–response curve of BK on the isolated rat uterus preparation in the absence and presence of the CFME of plant C. asiaticum; 1 ŽB., 5 Žv ., 10 Ž'. and 20 Ž`. mg mly1 . Each point represent the mean" S.E.M. of six observations. U Significant difference Ž p - 0.05. from values obtained from preparations without plant extract Žcontrol, I..
A.M. Samud et al.r Immunopharmacology 43 (1999) 311–316
Fig. 5. Dose–response curve of histamine on the isolated guineapig ileum preparation in the absence and presence of the CFME of plant C. asiaticum; 5 ŽB., 10 Žv ., 20 Ž'. and 50 Ž`. mg mly1 . Each point represent the mean"S.E.M. of six observations. U Significant difference Ž p- 0.05. from values obtained from preparations without plant extract Žcontrol, I..
cant change Ž p ) 0.05. in the oedema induced by carrageenan compared to the control group. 3.2. Isolated preparations Fig. 4 shows that the CFME at concentrations of 5, 10 and 20 mg mly1 caused a significant reduction Ž p - 0.05. of BK-induced contractions and depressed the maximal responses to 36.74%, 19.35% and 18.60%, respectively, on isolated rat uterus preparations as compared to the control values. Whereas Fig. 5 shows that CFME at concentrations of 20 and 50 mg mly1 caused significant Ž p - 0.05. concentration-related parallel displacement to the right when compared to histamine doses–response curve in the absence of CFME on isolated guinea-pig ileum preparations.
4. Discussion The present study indicated that the anti-inflammatory activity of methanol extract Ž50 mg kgy1 . was stronger than indomethacin in acute paw oedema induced by carrageenan in mice. On the contrary, the anti-inflammatory activity of CFME seemed to be equal to that of indomethacin. This may suggest that the principle components contained may not be the
315
same. It is of interest to state that there could be disintegration of some of the activity of chemical constituents during further fractionation. Furthermore, it might be suggestive of the presence of several active compounds. The work is in progress to determine the various active components in these extracts. It is well known that irritating compounds sometimes can cause pseudo inhibition of the oedema induced by carrageenan ŽOzaki, 1990.. Additionally, Damas et al. Ž1986. reported that carrageenan-induced oedema could be inhibited by the local application of the counter irritant in Brown Norway rats. In the present study, since the CFME was given orally, its anti-inflammatory effect could not have been due to its counter irritant property. Development of oedema induced by carrageenan is commonly correlated with the early exudative stage of inflammation, one of the important process of inflammatory pathology ŽOzaki, 1990.. Kinin-forming components and histamine have been known to play a major role in the pathophysiology of inflammatory diseases ŽSharma and Muhsin, 1990.. In the present experiments of BK-induced contraction of rat uterus, the maximum response was suppressed by CFME in a dose-dependent manner. This observation does not support the possibility that CFME agents are BK receptor antagonists, but may indicate non-specific inhibitors of contraction. Whereas, a dose–response curve of histamine in the guinea-pig ileum preparation was shifted to the right by the CFME, suggesting that a major part of the extract may be a histamine receptor antagonist. We observed that CFME has no significant effect on acetylcholine- and potassium chloride-induced contractile responses on isolated smooth muscle preparations Žunpublished data.. Recently, it is reported that BK is the prime mediator of initial as well as the later phase of carrageenan oedema ŽSharma, 1993; Sharma et al., 1998.. CFME inhibited the oedema induced by carrageenan and antagonised the contractile responses of BK and histamine in the present study. Therefore, the present findings suggest that C. asiaticum possessed an anti-inflammatory activity as suggested by its use in traditional medicine. The anti-inflammatory activity of this plant could not have been due to its anti-BK activities, but may be partly due to its anti-histaminic properties.
316
A.M. Samud et al.r Immunopharmacology 43 (1999) 311–316
Acknowledgements The research work has been supported by Ministry of Sciences and Technology of Malaysia ŽIRPA-RM7.. References Burkill, I.H., 1996. A dictionary of the economic products of the Malay peninsula. Ministry of Agriculture and Cooperative, Kuala Lumpur, pp. 690–691. Damas, J., Remacle-Volon, G., Deflandre, E., 1986. Further studies of the mechanism of counter irritation by turpentine. Naunyn-Schmiedeberg’s Arch. Pharmacol. 332, 196–200. Floyd, R.D., 1971. Techniques for evaluation of miscellaneous drug active on smooth muscle. In: Floyd, R.D. ŽEd.., Animal Experiments in Pharmacological Analysis. Charles Thomas Publisher, USA, pp. 223–3274. Ghosal, S., Shanthy, A., Kumar, A., Kumar, Y., 1985. Palmilycorine and lycoriside: acyloxy and acylglucosyloxy alkaloids from C. asiaticum. Phytochemistry 24, 2703–2706. Ochi, M., Otsuki, H., Nagao, K., 1976. The structure of Hamayne, a new alkaloid from C. asiaticum L. Var.japonicum Baker. Bull. Chem. Soc. Jpn. 49, 3363–3364.
Ozaki, Y., 1990. Anti-inflammatory effects of Curcuma xanthorrhiza Roxb, and its active principle. Chem. Pharm. Bull. 38, 1045–1048. Sharma, J.N., 1993. Therapeutic prospects of bradykinin receptor antagonists. Gen. Pharmacol. 24, 267–274. Sharma, J.N., Muhsin, S.M.S., 1990. Chemical mediators of inflammation with the emphasis on the kinin system. Exp. Pathol. 38, 73–96. Sharma, J.N., Zeitlin, I.J., 1977. Indomethacin in low concentration potentiates the actions of some spasmogens on the isolated oestrous rat uterus. J. Pharm. Pharmacol. 29, 316–317. Sharma, J.N., Yusof, A.P., Wirth, K.J., 1998. The kinin antagonist Hoe 140 reduced acute paw oedema in rats caused by carrageenan, bradykinin and kaolin. Inflammopharmacology 6, 9–17. Tharib, S., Veitch, G., EL-Migirab, S., 1983. Chromatographic Methods for Nature Product. University of Aston, Birmingham, pp. 24–36. Valenzuela, P., Guevara, R., Garcia, S., 1930. Lansium domesticum correa: a study of the chemistry of the rind and the pharmacodynamics of the resin obtained there. Nat. Appl. Bull. 1, 71–91. Winter, C., Risley, E., Nuss, G., 1962. Carrageenan induced oedema in hind paw of the rat as an assay for anti-inflammatory drugs. Proc. Soc. Biol. 111, 544–547.
Anti-inflammatory activity of Crinum asiaticum plant and its effect on bradykinin-induced contractions on isolated uterus Awatef M. Samud, M. Zaini Asmawi, Jagdish N. Sharma ) , Ahmad Pauzi M. Yusof Department of Pharmacology and Physiology, School of Pharmaceutical Sciences, UniÕersiti Sains Malaysia, 11800 Penang, Malaysia Accepted 3 June 1999
Abstract Crinum asiaticum Linn plant is used in Malaysia as a rheumatic remedy and to relieve local pain. In the present study, we examined the anti-inflammatory effects of this plant extract on carrageenan-induced hind paw oedema in mice. C. asiaticum was serially extracted with petroleum ether, followed by chloroform and lastly, methanol. The chloroform and methanol extracts of the plant given orally Ž50 mg kgy1 . caused significant Ž p - 0.05; n s 7. reduction in paw oedema but the petroleum ether extract did not induce significant effect Ž p ) 0.05. on paw oedema. The methanol extract was then dissolved in water and extracted consecutively with chloroform, ethyl acetate and butanol. The chloroform fraction of methanol extract ŽCFME. treatment Ž50 mg kgy1 . significantly reduced Ž p - 0.05; n s 7. the acute paw oedema. This may indicate that active anti-inflammatory compounds are present in the CFME. In an attempt to study the mechanism of action of its anti-inflammatory activity, the effects of CFME on BK- and histamine-induced contractions were investigated in isolated rat uterus and guinea-pig ileum preparations, respectively. It was found that CFME caused dose-dependent reduction Ž p - 0.05; n s 6. of the contractile response induced by BK and shifted the log dose–response curve of histamine to the right. The present findings suggest that C. asiaticum possessed an anti-inflammatory activity as suggested by its use in traditional medicine. The anti-inflammatory activity of this plant could not have been due to its anti-bradykinin activities as CFME non-specifically inhibited BK-induced contraction. It also suggest that CFME may contain compoundŽs. with anti-histaminic properties. The significance of these findings is discussed. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Anti-inflammatory activity; Carrageenan oedema; C. asiaticum; Bradykinin responses; Rat uterus
1. Introduction Crinum asiaticum Linn Žfamily: Amaryllidaceae. is a herbaceous plant of small to moderate size with greenish-feathery leaves. In Southeast Asian counAbbreÕiations: BK, Bradykinin; CMC, Carboxymethycellulose; CFME, Chloroform fraction of methanol extract of C. asiaticum plant ) Corresponding author. Fax: q60-604-6570017; e-mail: [email protected]
tries, C. asiaticum has a considerable medicinal reputation as a potent folk medicine in the treatment of injury and inflamed joints ŽBurkill, 1966.. In the Philippines, the leaves are prepared as emollient for external use to treat inflamed joints and sprains ŽValenzuela et al., 1930.. In early times, it is used in Indonesia and in Malay peninsula as an antidote for wounds from poisoned arrows. The poultice Žbroader leaves, oiled and heated. are applied to swollen joints and used to treat fever, headache and local pain ŽBurkill, 1966..
0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 9 . 0 0 1 3 2 - 0
312
A.M. Samud et al.r Immunopharmacology 43 (1999) 311–316
Although few pharmacological studies have been reported ŽOchi et al., 1976; Ghosal et al., 1985., this plant has not been subjected to systematic pharmacological anti-inflammatory evaluation to support its use. The present study, therefore, was carried out to evaluate the effect of C. asiaticum plant on an acute inflammatory model using carrageenan-induced oedema in mice. The BK and histamine are important inflammatory mediators. These mediators possess potent smooth muscle contracting properties. Hence, the effects of the plant extract on the contractile responses induced by BK and histamine on
isolated smooth muscle preparations were examined in an attempt to elucidate the involvement of these two mediators in the anti-inflammatory activity.
2. Materials and methods 2.1. Identification of plant material and preparation of extract Samples of fresh green leaves of C. asiaticum plant were collected in November, 1996 from Penang
Fig. 1. Fractionation of crude methanol extract of C. asiaticum.
A.M. Samud et al.r Immunopharmacology 43 (1999) 311–316
island, Malaysia. This plant had been identified and voucher specimens was deposited in the herbarium of the School of Biological Sciences, Universiti Sains Malaysia Žspecimen No. 10106.. C. asiaticum dried powdered leaves Ž950 g. were refluxed in Soxhlet’s apparatus with petroleum ether at 40–608C, chloroform at 45–558C and finally with methanol at 60–708C. The mark left after each extraction was completely dried from the previous solvent of extraction. The extracts were filtered and the filtrate were evaporated under reduced pressure ŽRotavapor, Buchi. The residues obtained were dried under vacuum at y1108C ŽVacuum system B-169, Buchi. for 10 h. The yield of the extraction procedure were 40.85 g Ž4.31%. for petroleum ether extract, 26.89 g Ž2.83%. for chloroform extract and 146.97 g Ž15.47%. for methanol extract. Using the maceration technique ŽTharib et al., 1983., the methanol extract was further fractionated according to the scheme shown in Fig. 1. The chloroform fraction which was obtained from the methanol extract is referred to hereafter as CFME.
313
2.3. Isolated preparations 2.3.1. Rat uterus Virgin rats Ž120–200 g; n s 6. were brought to artificial oestrus 18–22 h after a single S.C. injection of stilboestrol ŽSigma. 0.1 mg kgy1 solution in ethanol. Rat were killed and horns of uterus were suspended in De Jalon’s solution at 358C as described by Sharma and Zeitlin Ž1977.. BK doses-contractile responses were obtained in the presence and absence of CFME of C. asiaticum plant. CFME was tested at concentration of 1, 5, 10 and 20 mg mly1 . BK ŽSigma. was used at doses of 1, 2, 4 and 8 pg mly1 . 2.3.2. Guinea-pig ileum preparation Guinea-pig Ž250–350 gm; n s 6. were killed by a blow on the head and cutting the throat. The strip of ileum 2–3 cm was suspended in Tyrode’s solution at 378C ŽFloyd, 1971.. A dose contractile responses of histamine was obtained in the absence and presence of CFME of C. asiaticum plant. Histamine was used at doses of 0.1, 0.2, 0.4, 0.8 and 1.6 mg mly1 .
2.2. Carrageenan-induced oedema The procedure used to assess anti-inflammatory activity was based on the method used by Winter et al. Ž1962.. Food was withdrawn from the animals 24 h prior to the experiments, but were supplied with water ad libitum. Plant extract 50 mg kgy1 , indomethacin ŽSigma. 1 mg kgy1 suspended in 1% CMC as positive control and vehicle as negative control were administered orally to groups of mice Ž20–35 g; n s 7 each.. One hour later, oedema was induced by injecting in the right hind paw 0.02 ml carrageenan ŽSigma. 1% Ž100 mg carrageenan dissolved in 10 ml saline.. The pad thickness of hind paw was measured by micrometer Žmm; Mitutoyo. before and 3 h after S.C. injection of carrageenan. The anti-inflammatory activity of the plant extract and indomethacin to inhibit the swelling of the hind paw was calculated by the following formula: % inflammations Ž A y B . rB = 100 where A s measurement of pad thickness 3 h after carrageenan-induced oedema, B s initial measurement of pad thickness before carrageenan-induced oedema.
Fig. 2. Effect of oral administration of indomethacin Ž1 mg kgy1 ., petroleum ether extract Ž2 g kgy1 ., chloroform extract Ž50 mg kgy1 . and methanol extract Ž50 mg kgy1 . of plant C. asiaticum after 3 h of carrageenan-induced oedema in mice hind paw. Results are expressed as mean percentage of inflammation" S.E.M. U Significant difference Ž p- 0.05. from the control. Seven mice were used in each group.
314
A.M. Samud et al.r Immunopharmacology 43 (1999) 311–316
2.3.3. Statistical analysis Data were expressed as the mean " S.E.M. The data were evaluated for significance of difference using analysis of variance ŽANOVA.. The homogeneity of groups was verified by Duncan’s test at an alpha level equal to 5%.
3. Results
3.1. Effects of plant extracts on paw oedema
Fig. 3. Effect of oral administration of indomethacin Ž1 mg kgy1 .; n-butanol fraction Ž50 mg kgy1 ., chloroform fraction Ž50 mg kgy1 ., ethyl acetate fraction Ž50 mg kgy1 ., and water fraction Ž50 mg kgy1 . obtained from methanol extract of plant C. asiaticum after 3 h of carrageenan-induced oedema in mice hind paw. Results are expressed as mean percentage of inflammation" S.E.M. U Significant difference Ž p- 0.05. from the control. Seven mice were used in each group.
CFME was tested at doses of 5, 10, 20 and 50 mg mly1 .
Results in Fig. 2 shows that methanol and chloroform extracts Ž50 mg kgy1 . and indomethacin Ž1 mg kgy1 . caused significant Ž p - 0.05. inhibition of paw oedema Ž94.8%, 86.7% and 72.0%, respectively. as compared to the non-treated control group of mice. Petroleum ether extract, 2 gm kgy1 did not significantly Ž p ) 0.05. inhibit carrageenan-induced inflammation. Fig. 3 shows that CFME 50 mg kgy1 significantly inhibited carrageenan-induced oedema Ž p - 0.05.. The inhibitory potency was about the same as that of indomethacin 1 mg kgy1 . However, butanol, ethyl acetate and aqueous fractions Ž50 mg kgy1 . of the methanol extract did not cause signifi-
Fig. 4. Dose–response curve of BK on the isolated rat uterus preparation in the absence and presence of the CFME of plant C. asiaticum; 1 ŽB., 5 Žv ., 10 Ž'. and 20 Ž`. mg mly1 . Each point represent the mean" S.E.M. of six observations. U Significant difference Ž p - 0.05. from values obtained from preparations without plant extract Žcontrol, I..
A.M. Samud et al.r Immunopharmacology 43 (1999) 311–316
Fig. 5. Dose–response curve of histamine on the isolated guineapig ileum preparation in the absence and presence of the CFME of plant C. asiaticum; 5 ŽB., 10 Žv ., 20 Ž'. and 50 Ž`. mg mly1 . Each point represent the mean"S.E.M. of six observations. U Significant difference Ž p- 0.05. from values obtained from preparations without plant extract Žcontrol, I..
cant change Ž p ) 0.05. in the oedema induced by carrageenan compared to the control group. 3.2. Isolated preparations Fig. 4 shows that the CFME at concentrations of 5, 10 and 20 mg mly1 caused a significant reduction Ž p - 0.05. of BK-induced contractions and depressed the maximal responses to 36.74%, 19.35% and 18.60%, respectively, on isolated rat uterus preparations as compared to the control values. Whereas Fig. 5 shows that CFME at concentrations of 20 and 50 mg mly1 caused significant Ž p - 0.05. concentration-related parallel displacement to the right when compared to histamine doses–response curve in the absence of CFME on isolated guinea-pig ileum preparations.
4. Discussion The present study indicated that the anti-inflammatory activity of methanol extract Ž50 mg kgy1 . was stronger than indomethacin in acute paw oedema induced by carrageenan in mice. On the contrary, the anti-inflammatory activity of CFME seemed to be equal to that of indomethacin. This may suggest that the principle components contained may not be the
315
same. It is of interest to state that there could be disintegration of some of the activity of chemical constituents during further fractionation. Furthermore, it might be suggestive of the presence of several active compounds. The work is in progress to determine the various active components in these extracts. It is well known that irritating compounds sometimes can cause pseudo inhibition of the oedema induced by carrageenan ŽOzaki, 1990.. Additionally, Damas et al. Ž1986. reported that carrageenan-induced oedema could be inhibited by the local application of the counter irritant in Brown Norway rats. In the present study, since the CFME was given orally, its anti-inflammatory effect could not have been due to its counter irritant property. Development of oedema induced by carrageenan is commonly correlated with the early exudative stage of inflammation, one of the important process of inflammatory pathology ŽOzaki, 1990.. Kinin-forming components and histamine have been known to play a major role in the pathophysiology of inflammatory diseases ŽSharma and Muhsin, 1990.. In the present experiments of BK-induced contraction of rat uterus, the maximum response was suppressed by CFME in a dose-dependent manner. This observation does not support the possibility that CFME agents are BK receptor antagonists, but may indicate non-specific inhibitors of contraction. Whereas, a dose–response curve of histamine in the guinea-pig ileum preparation was shifted to the right by the CFME, suggesting that a major part of the extract may be a histamine receptor antagonist. We observed that CFME has no significant effect on acetylcholine- and potassium chloride-induced contractile responses on isolated smooth muscle preparations Žunpublished data.. Recently, it is reported that BK is the prime mediator of initial as well as the later phase of carrageenan oedema ŽSharma, 1993; Sharma et al., 1998.. CFME inhibited the oedema induced by carrageenan and antagonised the contractile responses of BK and histamine in the present study. Therefore, the present findings suggest that C. asiaticum possessed an anti-inflammatory activity as suggested by its use in traditional medicine. The anti-inflammatory activity of this plant could not have been due to its anti-BK activities, but may be partly due to its anti-histaminic properties.
316
A.M. Samud et al.r Immunopharmacology 43 (1999) 311–316
Acknowledgements The research work has been supported by Ministry of Sciences and Technology of Malaysia ŽIRPA-RM7.. References Burkill, I.H., 1996. A dictionary of the economic products of the Malay peninsula. Ministry of Agriculture and Cooperative, Kuala Lumpur, pp. 690–691. Damas, J., Remacle-Volon, G., Deflandre, E., 1986. Further studies of the mechanism of counter irritation by turpentine. Naunyn-Schmiedeberg’s Arch. Pharmacol. 332, 196–200. Floyd, R.D., 1971. Techniques for evaluation of miscellaneous drug active on smooth muscle. In: Floyd, R.D. ŽEd.., Animal Experiments in Pharmacological Analysis. Charles Thomas Publisher, USA, pp. 223–3274. Ghosal, S., Shanthy, A., Kumar, A., Kumar, Y., 1985. Palmilycorine and lycoriside: acyloxy and acylglucosyloxy alkaloids from C. asiaticum. Phytochemistry 24, 2703–2706. Ochi, M., Otsuki, H., Nagao, K., 1976. The structure of Hamayne, a new alkaloid from C. asiaticum L. Var.japonicum Baker. Bull. Chem. Soc. Jpn. 49, 3363–3364.
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