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Gossypin-induced analgesia in mice
European Journal of Pharmacology, 98 (1984) 289-291
289
Elsevier Short communication
G O S S Y P I N - I N D U C E D ANALGESIA IN MICE SUBRAMANIAN VISWANATHAN*, PANCHANATHAN THIRUGNANA SAMBANTHAM, KANNAPPA REDDY and LALITHA KAMESWARAN Medicinal Chemistry Research Centre, Institute of Pharmacology, Madras Medical College, Madras 600 003, India
Received 13 December 1983, accepted 27 December 1983
S. VISWANATHAN, P.T. SAMBANTHAM, K. REDDY and L. KAMESWARAN, Gossypin-induced analgesia in mice, European J. Pharmacol. 98 (1984) 289-291.
A flavonoid, gossypin, was evaluated for its analgesic action by using acetic acid-induced writhing in mice and was compared with morphine. Gossypin inhibited writhing in a dose-dependent manner. This action was antagonised by naloxone. The pA 2 values for morphine-naloxone and gossypin-naloxone were almost identical suggesting a definite involvement of opiate receptors in the analgesic action of gossypin. Gossypin
Analgesia
Opiate receptor
1. Introduction
Flavonoids (vitamin P compounds) have a role in the maintenance of the integrity of the vessel wall (Clark and Geismann, 1949) and exhibit several beneficial actions such as anti-hepatotoxic, anti-inflammatory, anti-ulcer activities and protection against galactosemic cataracts (Varma et al., 1977). Interestingly Ramaswamy et al. (1980) reported the analgesic activity of O-fl-hydroxyethyl rutoside (HR) and suggested that adrenergic mechanism may be involved in effecting analgesia. In the present study, an attempt was made to investigate the analgesic activity of gossypin (Bioorganics, Madras), another flavonoid and the possible mechanism of this action.
2. Materials and methods
Male albino mice (20-25 g) were used in this study. The analgesia was assessed by employing the writhing test. Acetic acid (0.6%, 10 ml/kg) was injected i.p. (Koster et al., 1959) and the number * To whom all correspondenceshould be addressed. 0014-2999/84/$03.00 © 1984 ElsevierSciencePublishers B.V.
of writhings for 15 min after acetic acid injection was noted. Gossypin was administered in varying doses (12.5, 25, 50 and 100 m g / k g i.p.) 60 min prior to acetic acid challenge. For comparison, the effect of morphine sulphate (Govt. opium and alkaloids, Ghazipur) was studied by injecting (0.125, 0.25, 0.5, 1 and 2 m g / k g s.c.) 30 rain prior to acetic acid challenge. The involvement of opiate receptors was assessed by pretreating a separate group of mice with naloxone (Endo labs) (0.01, 0.05 and 0.1 m g / k g s.c.) 10 rain prior to gossypin or morphine sulphate administration. The percentage inhibition of writhing by either gossypin or morphine was plotted against the log dose. The dose which produced 50% inhibition of writhing was determined (EDs0). The ED50 values were also obtained in the animals pretreated with naloxone in three varying concentrations and the apparent pA 2 values were calculated for gossypinnaloxone and morphine-naloxone using the formula, log(dose ratio - 1) = pA 2 + log B, where B is the molar concentration of the antagonist (Arunlakshna and Schild 1959). The results were analysed using Student's t-test.
290
3. Resets
4. Discussion
G o s s y p i n in all the doses tested significantly r e d u c e d the n u m b e r of writhings i n d u c e d b y acetic acid. The effect was d o s e - d e p e n d e n t a n d the maxim u m effect was o b s e r v e d at 100 mg (table 1). M o r p h i n e also d e c r e a s e d the n u m b e r of writhings in a d o s e - d e p e n d e n t m a n n e r (table 1). T h e effect of gossypin was significantly a n t a g o n i s e d b y pret r e a t m e n t with naloxine similarly to w h a t was o b s e r v e d for m o r p h i n e (table 1), T h e r e was a p a r a l l e l shift of the d o s e - r e s p o n s e curve of gossypin to the right in the presence of naloxone indicating competitive antagonism. A similar parallel shift of the d o s e - r e s p o n s e curve to the right for m o r p h i n e was o b s e r v e d in the presence of naloxone. T h e EDs0 values for gossypin a n d m o r p h i n e a n d their changes after n a l o x o n e are shown in table 1. T h e EDs0 of gossypin was m u c h higher t h a n that of m o r p h i n e . T h e a p p a r e n t p A 2 values for g o s s y p i n - n a l o x o n e a n d m o r p h i n e - n a l o x o n e were f o u n d to be a l m o s t identical; i.e. 7.52 +__0.21 a n d 7.38 + 0.12 ( m e a n + S.E.) respectively.
T h e results of the present s t u d y show a signific a n t d o s e - d e p e n d e n t analgesic activity for gossyp i n as evidenced b y the decrease in the n u m b e r of acetic a c i d - i n d u c e d writhings. Similar to the EDs0 for other beneficial actions (Parmar, 1977) the EDs0 of gossypin with respect to the analgesic effect was f o u n d to be higher. However, the effect was c o m p a r a b l e to that of m o r p h i n e . It is also essential to note that the LDs0 for most of the flavonoids including gossypin is greater than 25 g / k g . H e n c e the EDs0 for the analgesic effect (17.38 m g / k g ) is well b e l o w the toxic dose. T h e analgesic effect of gossypin was a n t a g o n i s e d b y naloxone, an o p i a t e receptor antagonist, indic a t i n g a definite i n v o l v e m e n t of o p i a t e receptors. T h e c o n c e p t of p A 2 for assessing competitive a n t a g o n i s m (Schild, 1947) has been e m p l o y e d to c h a r a c t e r i s e analgesic receptors in vivo (Smits and T a k e m o r i , 1970; H y a s h i a n d T a k e m o r i , 1971). A c c o r d i n g to the p A 2 concept, if the drugs interact with a similar t y p e of receptor, similar p A 2 values will be o b t a i n e d ( A r u n l a k s h n a a n d Schild, 1959).
TABLE 1 Effect of gossypin and morphine on acetic acid writhing in the absence and presence of naloxone. Each value represents the mean ± S.E.M. of at least six experiments. Drug mg/kg
Numberofwrithings Without naloxone
With naloxone (mg/kg) 0.01
Control Gossypin 12.5 25 50 100 Morphine 0.125 0,25 0.5 1.0 2.0
0.05
31.4±2.5
0.1
30.8±3.2
19.0±2.6 a 12.0±2.0 a 5.0±2.0" 0.0 a
26.5±0.9 22.5±2.7 16.6±1.2 8.0±1.9
h h b b
19.2±0.9 a 15.5±3.0 a 12.0±4.9" 6.8±2.8 a 2.2±1.0 a
26.1±1.9 b 19.5±1.1 b 15.3±1.0 9.8±2.0 3.5±1.6
28.6±1.36 b 21.7±1.6 b 16.2±3.6 b 11.2±1.9 b
29.3±0.42 h 24.0±3.4 b 19.2±6.0 b 13.8±0.4 b
26.5±2.2 b 21.8±1.2 b 17.1±1.0 13.0±1.2 ~ 8.8±2.7 b
26.8±0.6 h 23.6±1.1 b 20.2±2.1 ~ 17.8±2.0 b 13.8±0.9 h
a p < 0.001 with respect to the control value, b p < 0.001 with respect to the value obtained without naloxone pretreatment.
291
By employing three different analgesic assays, viz. tail-flick, hot plate and chemically induced writhing, Hyashi and Takemori (1971) found that the pA 2 values for the morphine-naloxone combination were almost similar irrespective of the test procedure used. They also found that the EDs0 value for morphine in the writhing test was the lowest (0.33 mg) as compared to other two assays. In the present study a similar EDs0 value (0.24 mg) for morphine was obtained. Since r~aloxone antagonised the gossypin-induced analgesic effect it was of interest to study the pA 2 for gossypinnaloxone and compare it with that of the morphine-naloxone combination. The results revealed almost identical pA 2 values for gossypin and morphine and hence it can be suggested that the opiate receptors do participate in the gossypin-induced analgesic action. Various neurotransmitters e.g. 5-hydroxytryptamine, acetylcholine or norepinephrine are implicated in effecting opiate analgesia. Such a possibility remains to be investigated for the analgesic action of gossypin.
Acknowledgements The Authors express their thanks to the Convenor, Medicinal Chemistry Research Centre and the Dean, Madras Medical College, for the facilities provided.
References Arunlakshna, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Br. J. Pharmacol. 14, 48. Clark, W.G. and T.A. Geismann, 1949, Potentiation of effects of epinephrine by flavonoid (vitamin P like) compounds. Relation of structure to activity, J. Pharmacol. Exp. Ther. 95, 363. Hyashi, G. and A.E. Takemori, 1971, The type of analgesic receptor interaction involved in certain analgesic assays, European J. Pharmacol. 16, 63. Koster, R.M., M. Anderson and E.J. De Beer, 1959, Acetic acid for analgesic screening, Fed. Proc. 18, 412. Parmar, N.S., 1977, A pharmacological study on the effect of some bioflavonoids on experimentally induced inflammation, increased vascular permeability, gastric ulcers and galactosemic cataracts, Ph.D. thesis, Madras University. Ramaswamy, S., N. Padmanabha Pillai, V. Gopalakrishnan and N.S. Parmar, 1980, Analgesic activity of a bioflavonoid in mice, Ind. J. Pharmacol. 12(1), 33. Schild, H.O., 1947, PA. A new scale for the measurement of drug antagonism, Br. J. Pharmacol. 2, 189. Smits, S.E. and A.E. Takemori, 1970, Quantitative studies on the antagonism by naloxone of some narcotic and narcotic antagonist analgesics, Br. J. Pharmacol. 39, 627. Varma, S.D., A. Mizuno and J.H. Kinoshita, 1977, Diabetic Cataracts and flavonoids, Science 195 (4274), 205.
289
Elsevier Short communication
G O S S Y P I N - I N D U C E D ANALGESIA IN MICE SUBRAMANIAN VISWANATHAN*, PANCHANATHAN THIRUGNANA SAMBANTHAM, KANNAPPA REDDY and LALITHA KAMESWARAN Medicinal Chemistry Research Centre, Institute of Pharmacology, Madras Medical College, Madras 600 003, India
Received 13 December 1983, accepted 27 December 1983
S. VISWANATHAN, P.T. SAMBANTHAM, K. REDDY and L. KAMESWARAN, Gossypin-induced analgesia in mice, European J. Pharmacol. 98 (1984) 289-291.
A flavonoid, gossypin, was evaluated for its analgesic action by using acetic acid-induced writhing in mice and was compared with morphine. Gossypin inhibited writhing in a dose-dependent manner. This action was antagonised by naloxone. The pA 2 values for morphine-naloxone and gossypin-naloxone were almost identical suggesting a definite involvement of opiate receptors in the analgesic action of gossypin. Gossypin
Analgesia
Opiate receptor
1. Introduction
Flavonoids (vitamin P compounds) have a role in the maintenance of the integrity of the vessel wall (Clark and Geismann, 1949) and exhibit several beneficial actions such as anti-hepatotoxic, anti-inflammatory, anti-ulcer activities and protection against galactosemic cataracts (Varma et al., 1977). Interestingly Ramaswamy et al. (1980) reported the analgesic activity of O-fl-hydroxyethyl rutoside (HR) and suggested that adrenergic mechanism may be involved in effecting analgesia. In the present study, an attempt was made to investigate the analgesic activity of gossypin (Bioorganics, Madras), another flavonoid and the possible mechanism of this action.
2. Materials and methods
Male albino mice (20-25 g) were used in this study. The analgesia was assessed by employing the writhing test. Acetic acid (0.6%, 10 ml/kg) was injected i.p. (Koster et al., 1959) and the number * To whom all correspondenceshould be addressed. 0014-2999/84/$03.00 © 1984 ElsevierSciencePublishers B.V.
of writhings for 15 min after acetic acid injection was noted. Gossypin was administered in varying doses (12.5, 25, 50 and 100 m g / k g i.p.) 60 min prior to acetic acid challenge. For comparison, the effect of morphine sulphate (Govt. opium and alkaloids, Ghazipur) was studied by injecting (0.125, 0.25, 0.5, 1 and 2 m g / k g s.c.) 30 rain prior to acetic acid challenge. The involvement of opiate receptors was assessed by pretreating a separate group of mice with naloxone (Endo labs) (0.01, 0.05 and 0.1 m g / k g s.c.) 10 rain prior to gossypin or morphine sulphate administration. The percentage inhibition of writhing by either gossypin or morphine was plotted against the log dose. The dose which produced 50% inhibition of writhing was determined (EDs0). The ED50 values were also obtained in the animals pretreated with naloxone in three varying concentrations and the apparent pA 2 values were calculated for gossypinnaloxone and morphine-naloxone using the formula, log(dose ratio - 1) = pA 2 + log B, where B is the molar concentration of the antagonist (Arunlakshna and Schild 1959). The results were analysed using Student's t-test.
290
3. Resets
4. Discussion
G o s s y p i n in all the doses tested significantly r e d u c e d the n u m b e r of writhings i n d u c e d b y acetic acid. The effect was d o s e - d e p e n d e n t a n d the maxim u m effect was o b s e r v e d at 100 mg (table 1). M o r p h i n e also d e c r e a s e d the n u m b e r of writhings in a d o s e - d e p e n d e n t m a n n e r (table 1). T h e effect of gossypin was significantly a n t a g o n i s e d b y pret r e a t m e n t with naloxine similarly to w h a t was o b s e r v e d for m o r p h i n e (table 1), T h e r e was a p a r a l l e l shift of the d o s e - r e s p o n s e curve of gossypin to the right in the presence of naloxone indicating competitive antagonism. A similar parallel shift of the d o s e - r e s p o n s e curve to the right for m o r p h i n e was o b s e r v e d in the presence of naloxone. T h e EDs0 values for gossypin a n d m o r p h i n e a n d their changes after n a l o x o n e are shown in table 1. T h e EDs0 of gossypin was m u c h higher t h a n that of m o r p h i n e . T h e a p p a r e n t p A 2 values for g o s s y p i n - n a l o x o n e a n d m o r p h i n e - n a l o x o n e were f o u n d to be a l m o s t identical; i.e. 7.52 +__0.21 a n d 7.38 + 0.12 ( m e a n + S.E.) respectively.
T h e results of the present s t u d y show a signific a n t d o s e - d e p e n d e n t analgesic activity for gossyp i n as evidenced b y the decrease in the n u m b e r of acetic a c i d - i n d u c e d writhings. Similar to the EDs0 for other beneficial actions (Parmar, 1977) the EDs0 of gossypin with respect to the analgesic effect was f o u n d to be higher. However, the effect was c o m p a r a b l e to that of m o r p h i n e . It is also essential to note that the LDs0 for most of the flavonoids including gossypin is greater than 25 g / k g . H e n c e the EDs0 for the analgesic effect (17.38 m g / k g ) is well b e l o w the toxic dose. T h e analgesic effect of gossypin was a n t a g o n i s e d b y naloxone, an o p i a t e receptor antagonist, indic a t i n g a definite i n v o l v e m e n t of o p i a t e receptors. T h e c o n c e p t of p A 2 for assessing competitive a n t a g o n i s m (Schild, 1947) has been e m p l o y e d to c h a r a c t e r i s e analgesic receptors in vivo (Smits and T a k e m o r i , 1970; H y a s h i a n d T a k e m o r i , 1971). A c c o r d i n g to the p A 2 concept, if the drugs interact with a similar t y p e of receptor, similar p A 2 values will be o b t a i n e d ( A r u n l a k s h n a a n d Schild, 1959).
TABLE 1 Effect of gossypin and morphine on acetic acid writhing in the absence and presence of naloxone. Each value represents the mean ± S.E.M. of at least six experiments. Drug mg/kg
Numberofwrithings Without naloxone
With naloxone (mg/kg) 0.01
Control Gossypin 12.5 25 50 100 Morphine 0.125 0,25 0.5 1.0 2.0
0.05
31.4±2.5
0.1
30.8±3.2
19.0±2.6 a 12.0±2.0 a 5.0±2.0" 0.0 a
26.5±0.9 22.5±2.7 16.6±1.2 8.0±1.9
h h b b
19.2±0.9 a 15.5±3.0 a 12.0±4.9" 6.8±2.8 a 2.2±1.0 a
26.1±1.9 b 19.5±1.1 b 15.3±1.0 9.8±2.0 3.5±1.6
28.6±1.36 b 21.7±1.6 b 16.2±3.6 b 11.2±1.9 b
29.3±0.42 h 24.0±3.4 b 19.2±6.0 b 13.8±0.4 b
26.5±2.2 b 21.8±1.2 b 17.1±1.0 13.0±1.2 ~ 8.8±2.7 b
26.8±0.6 h 23.6±1.1 b 20.2±2.1 ~ 17.8±2.0 b 13.8±0.9 h
a p < 0.001 with respect to the control value, b p < 0.001 with respect to the value obtained without naloxone pretreatment.
291
By employing three different analgesic assays, viz. tail-flick, hot plate and chemically induced writhing, Hyashi and Takemori (1971) found that the pA 2 values for the morphine-naloxone combination were almost similar irrespective of the test procedure used. They also found that the EDs0 value for morphine in the writhing test was the lowest (0.33 mg) as compared to other two assays. In the present study a similar EDs0 value (0.24 mg) for morphine was obtained. Since r~aloxone antagonised the gossypin-induced analgesic effect it was of interest to study the pA 2 for gossypinnaloxone and compare it with that of the morphine-naloxone combination. The results revealed almost identical pA 2 values for gossypin and morphine and hence it can be suggested that the opiate receptors do participate in the gossypin-induced analgesic action. Various neurotransmitters e.g. 5-hydroxytryptamine, acetylcholine or norepinephrine are implicated in effecting opiate analgesia. Such a possibility remains to be investigated for the analgesic action of gossypin.
Acknowledgements The Authors express their thanks to the Convenor, Medicinal Chemistry Research Centre and the Dean, Madras Medical College, for the facilities provided.
References Arunlakshna, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Br. J. Pharmacol. 14, 48. Clark, W.G. and T.A. Geismann, 1949, Potentiation of effects of epinephrine by flavonoid (vitamin P like) compounds. Relation of structure to activity, J. Pharmacol. Exp. Ther. 95, 363. Hyashi, G. and A.E. Takemori, 1971, The type of analgesic receptor interaction involved in certain analgesic assays, European J. Pharmacol. 16, 63. Koster, R.M., M. Anderson and E.J. De Beer, 1959, Acetic acid for analgesic screening, Fed. Proc. 18, 412. Parmar, N.S., 1977, A pharmacological study on the effect of some bioflavonoids on experimentally induced inflammation, increased vascular permeability, gastric ulcers and galactosemic cataracts, Ph.D. thesis, Madras University. Ramaswamy, S., N. Padmanabha Pillai, V. Gopalakrishnan and N.S. Parmar, 1980, Analgesic activity of a bioflavonoid in mice, Ind. J. Pharmacol. 12(1), 33. Schild, H.O., 1947, PA. A new scale for the measurement of drug antagonism, Br. J. Pharmacol. 2, 189. Smits, S.E. and A.E. Takemori, 1970, Quantitative studies on the antagonism by naloxone of some narcotic and narcotic antagonist analgesics, Br. J. Pharmacol. 39, 627. Varma, S.D., A. Mizuno and J.H. Kinoshita, 1977, Diabetic Cataracts and flavonoids, Science 195 (4274), 205.