Evaluation and comparison of antinociceptive activity of aspartame with sucrose

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1 Pharmacological Reports 2012, 64, ISSN Copyright 2012 by Institute of Pharmacology Polish Academy of Sciences Evaluation and comparison of antinociceptive activity of aspartame with sucrose Seema Rani, Mahesh C. Gupta Department of Pharmacology, Pt. B.D. Sharma PGIMS, Rohtak, Haryana, India Correspondence: Seema Rani, Abstract: Background: Artificial sweeteners are low-calorie substances used to sweeten a wide variety of foods. At present they are used increasingly not only by diabetics, but also by the general public as a mean of controlling the weight. This study was carried out to evaluate and compare antinociceptive activity of the artificial sweeteners, aspartame and sucrose and to study the mechanisms involved in this analgesic activity. Methods: Forty eight white albino Wistar rats were divided into two groups of 24 rats each. Group 1 received sucrose and group 2 received aspartame solution ad libitum for 14 days as their only source of liquid. On 14 th day, both groups of rats were divided into 3 subgroups having 8 rats each. Group Ia and IIa served as control. Group Ib and IIb were given naloxone and Ic and IIc received ketanserin, the opioid and serotonergic receptor antagonists, respectively. Results: Tail withdrawal latencies (tail flick analgesiometer) and paw licking/jumping latencies (Eddy s hot plate method) were increased significantly in both aspartame and sucrose group. The analgesia produced by aspartame was comparable with sucrose. The opioid receptor antagonist naloxone and the 5-HT 2A/2C serotonergic receptor antagonist ketanserin partly reversed the antinociceptive effect of these sweeteners. Conclusions: Thus, the artificial sweetening agent aspartame showed antinociceptive activity like sucrose in rats. Reduction in antinociceptive activity of aspartame and sucrose by opioid and serotoninergic antagonists demonstrate the involvement of both opioid and serotonergic system. Key words: aspartame, ketanserin, naloxone, sucrose, sweet-substance-induced antinociception Introduction Artificial sweeteners are low-calorie substances which have been used to sweeten a wide variety of foods with the intent of reducing intake of calories. These substances are very popular and are being increasingly used not only by diabetics, but also by the general public as a mean of controlling the weight. Aspartame, one such noncaloric sweetener, has been in wide use with many foods and beverages. Since these sweeteners are being so commonly used by the general population, it may be essential to evaluate their other pharmacological properties. One such pharmacological effect which has been recently studied is their antinociceptive activity [10, 16, 20, 21]. So it may be hypothesized that in addition to their role in weight control, these may be of help in attenuation of pain, especially associated with diabetic neuropathy, where these sweeteners are frequently used to reduce the intake of sugar. Whether all the sweeteners used commonly may have an antinociceptive activity or not is not known. The mechanisms underlying this Pharmacological Reports, 2012, 64,

2 antinociception action are also not very clear. Saccharin, one such sweetener, taken by rats for relatively long periods of time show an increase in the latency of paw withdrawal in the hot-plate test [4]. It was also demonstrated that consumption of concentrated sucrose solution seems to reduce crying and other pain related behavior in healthy normal babies [8]. It has also been shown that sweet substances may potentiate the analgesia of opiates [19]. Sweet palatable substances such as sucrose and polycose potentiate the morphine-induced analgesia, suggesting that interaction of these substances with opioid system modifies the sensitivity to pain stimuli [7]. There is evidence that endogenous opioid receptors may be involved in antinociception induced by the sweeteners [18]. It was also reported the involvement of serotonergic mechanism in sweet substance-induced antinociception as methylsergide and ketanserin antagonises the sweet-substance-induced analgesia. [18]. Since some neural pathways and some neurotransmitters play an important role in the complex modulation of pain transmission, the investigation of these modulatory mechanisms may have important implications for pain treatment. The use of different models for measuring pain is important because analgesic effect may be due to one or more mechanisms. For this reason, tail-flick is used for spinal reflex [25] and Eddy s hot plate test is used for measuring supraspinal pain-related mechanisms [14]. Therefore, this study was carried out to evaluate the antinociceptive activity of artificial sweeteners, aspartame and sucrose and also to explore the role of opioid and serotoninergic systems in such antinociceptive activity. Materials and Methods Animals Wistar albino rats weighing g with access to food and water ad libtum were used. These animals were housed eight per cage. The study was conducted in Department of Pharmacology, Pt. B.D. Sharma PGIMS, Rohtak. The protocol was approved by Institutional Animal Committee (IAEC) and all experiments were performed in accordance with the recommendations of guidelines for care and use of laboratory animals. Drugs The drugs used were aspartame in a dose of 1.6 g/l [17] and sucrose in a dose of 250 g/l, [20]; naloxone, 1 mg/kg [20] and ketanserin, 1 mg/kg [1] were used as antagonists. Aspartame and sucrose were dissolved in tap water just prior to administration. These were given orally for 14 days. Naloxone and ketanserin were given intraperitoneally on 14 th day. Experimental methods Analgesia was evaluated using tail flick test (tail withdrawl from radiant heat) by Techno-analgesiometer and hot plate (paw licking or jumping from the hot plate at 55 C) method by using Eddy s hot plate analgesiometer. Tail-flick test The tail-flick test was used in rats to elicit a spinal tail flick response to noxious thermal stimuli. The test was performed with the tail-flick model using analgesiometer. Each rat was gently held with one hand and the distal half of its tail was positioned on the source of radiant heat. The tail-flick response was elicited by applying radiant heat to the ventral surface of the tail. The time elapsed till the animal flicked its tail was determined (usual response 3 4 s). A 10 s cut off latency was kept to prevent damage to tail. Hot-plate test The hot-plate test was performed using an electronically controlled hot plate heated to 55 ± 0.1 C. Each rat was placed unrestrained on the hot plate until either paw licking or jumping occurred. A cut of time was kept at 15 s. Forty eight male Wistar albino rats were divided into two groups of twenty four rats each and were kept in six cages. Group I received sucrose solution 250 g/l and group II received 1.6 g/l aspartame solution orally ad libitum, respectively, for 14 days as their only source of liquid. Both the solutions were prepared in tap water. In both groups, pain threshold baseline (tail-flick latencies and paw licking or jumping responses) were recorded on day 1 and again on day 14. On 14 th day each group of rats was divided into 3 subgroups having 8 rats each. Group Ia and IIa served as control. Group Ib and IIb were given na- 294 Pharmacological Reports, 2012, 64,

3 Antinociceptive activity of aspartame and sucrose Seema Rani and Mahesh C. Gupta Tab. 1. Antinociceptive activity of sucrose and aspartame after opioid and sertoninergic antagonist. Effect reported 10 min after administration of naloxone (1 mg/kg) and ketansein (1 mg/kg) intraperitoneally. Data are reported as the mean ± SEM for 24 rats in each group and analyzed by two-way ANOVA followed by Turkey multiple comparison test. * p < 0.05 considered as significant Experimental model Day 14 After naloxone p value After ketanserin p value Sucrose Aspartame Sucrose Aspartame Sucrose Aspartame Tail flick latency 3.22 ± ± ± ± * Eddy s hot plate method 3.71 ± ± ± ± * 2.88 ± ± * 3.38 ± ± * * p value is for sucrose; ** p value is for aspartame loxone (1 mg/kg) and Ic and IIc received ketanserin (1 mg/kg) intraperitoneally. All rats were subjected to nociceptive testing using the tail flick test and Eddy s hot plate method 10 min after giving naloxone and ketanserin. The values of tail flick and paw licking were recorded as the mean (s) ± SEM. The latency values were statistically analyzed by analysis of variance (ANOVA) followed by Tukey s post-hoc test. Results Tail flick latency In tail flick method, tail flick latencies in group I at baseline, on day 1 and day 14 th were 2.88 ± 0.16, 2.83 ± 0.16 and 3.22 ± 0.11 s, respectively, while in group II, the values were 2.84 ± 0.16, 2.94 ± 0.13, and 3.22 ± 0.06 s, respectively. The tail flick latencies in both groups did not reflect significant change on day 1 (p > 0.05 in both groups) while these values were increased significantly on day 14 th [F (2, 69) = 9.09 in group I, F (2, 69) = in group II, p < 0.05 in both groups]. This signifies that both aspartame and sucrose produce significant antinociceptive effect on day14 th (Fig. 1). Both aspartame and sucrose were almost equally effective in producing this analgesic effect. After administration of naloxone, the mean tail flick latency on day 14 th was 2.83 ± 0.14 s in group I and 2.94 ± 0.13 s in group II. It was found a statistically significant attenuation of antinociceptive effect after administrations of naloxone in both groups I and II [F (5, 47) = 3.39, p < 0.05]. Similarly, the latency values after administration of ketanserin on day 14 th were 2.88 ± 0.14 s and 2.88 ± 0.10 s in group I and II, respectively (p < 0.05), which show a significant attenuation of antinociceptive effect. (Tab. 1). Paw licking/jumping latencies In Eddy s hot plate method, baseline, day 1 and day 14 th paw licking/jumping latencies were 3.49 ± 0.07, 3.49 ± 0.06 and 3.71 ± 0.10 s respectively, in group I while in group II the values were 3.5 ± 0.18, 3.49 ± 0.07 and 3.88 ± 0.11 s, respectively. Paw licking/jumping latencies were also reflecting no change on day 1 (p > 0.05 in both groups) while these increased on day 14 th [F (2, 69) = 7.96 in group I, F (2, 69) = in group II, p < 0.05 in both groups] (Fig. 2). After administration of naloxone, the mean paw licking/jumping latencies on day 14 th were 3.44 ± Time (s) sucrose aspartame Baseline Day 1 Day 14 Time intervals Fig. 1. Antinociceptive activity of sucrose and aspartame by tail flick method. Tail flick latencies were recorded at different time intervals (on day 0, 1 and 14 th after giving sucrose and aspartame to two different groups of rats) by tail flick method. Data are reported as the mean ± SEM for 24 rats in each group and analyzed by repeated measure ANOVA followed by post-hoc analysis Tukey multiple comparison test; * p < 0.05 considered as significant Pharmacological Reports, 2012, 64,

4 Time (s) s in group I and 3.38 ± 0.10 s in group II. There was an attenuation of antinociceptive effect after administration of naloxone in both groups I and II, which was statistically significant [F (5, 47) = 7.18, p < 0.05]. Similarly, the latency values, after administration of ketanserin were 3.38 ± 0.10 and 3.38 ± 0.05 s in group I and II, respectively, which were statistically significant (Tab. 1). Naloxoe and ketanserin both partly reversed the antinociceptive effect of both sweeteners. Discussion sucrose aspartame Baseline Day 1 Day 14 Time intervals Fig. 2. Antinociceptive activity of sucrose and aspartame by Eddy s hot plate method. Paw licking/jumping response were recorded at different time intervals (on day 0, 1 and 14 th after giving sucrose and aspartame to different rats) by Eddy s hot plate method. Data are reported as the mean ± SEM for 24 rats in each group and analyzed by repeated measure ANOVA followed by Tukey multiple comparison test; * p < 0.05 considered as significant The present study was conducted to evaluate the analgesic effect of two commonly used sweeteners aspartame and sucrose and to explore the mechanism of action of such antinociceptive effect. The results indicate that both the sweeteners produce a significant analgesic effect when regularly ingested over a period of 14 days. Such an antinociceptive effect with a variety of sweeteners like sucrose, glucose, fructose and lactose has already been demonstrated in several earlier studies [2, 4, 6, 20]. In fact, saccharin, an artificial sweetener extensively used in the past, has also been demonstrated to have an antinociceptive effect [4, 13, 15]. The antinociceptive effect with sucrose and aspartame was demonstrated in both experimental models, i.e., tail flick analgesiometer and Eddy s hot plate induced nociception. Tail flick-induced nociception has been considered to involve spinal mechanisms while Eddy s hot plate-induced nociception involves the supraspinal mechanisms [25]. These sweeteners produce antinociceptive effect in these both experimental models, indicating that this antinociceptive effect may involve both spinal and supraspinal mechanisms. A number of mechanisms have been hypothesized for the antinociceptive effect produced by these artificial sweeteners and the involvement of endogenous opioids, serotonergic mechanisms, muscarinic and nicotinic cholinergic receptors have been earlier postulated [10, 18, 20]. Studies also showed the involvement of dorsal raphe nucleus and locus coeruleus in the elaboration of the sweet substance-induced antinociception [12, 16]. This study evaluated the involvement of opioid and serotonergic mechanisms by using the appropriate antagonists, naloxone and ketanserin. Both naloxone and ketanserin antagonized the antinociceptive effect of both sucrose and aspartame. Naloxone was found to be more effective on spinal mechanisms while ketanserin was seen to be more effective on supraspinal mechanisms. Involvement of endogenous opioids for such analgesia has earlier been demonstrated [5] where reversal of analgesia was caused by naltrexone or naloxone [6, 20] and serotonin seems to be involved in the modulation of this response [18]. An increase in the level of -endorphins in the hypothalamus of rats by sweetening agents has also been demonstrated [3]. However, the effect of endogenous opioids may be also correlated with serotonin release from the spinal cord in diabetic rats [21, 22]. Results of recent studies have also shown evidence that the pain occurring in diabetic neuropathy can be decreased by indirect activation of central 5-HT 1 and 5-HT 2 receptors [23]. In fact, it was already shown that hydroxytryptaminergic antinociceptive pathways are hardly affected by diabetes [24]. In the present study, we have to consider the involvement of other opioid receptors, such as µ 2, or as naloxone acts on all subtypes of opioid receptors. Although ketanserin is more selective to 5-HT 2A/2C receptors, the involvement of other serotonergic receptors subtypes, such as 5-HT 1A, 5-HT 1D and 5-HT 3, cannot be discarded. In addition, taking into account the effects of ketanserin in 1 -adrenergic receptors, it is possible that the noradrenergic pathways also play some role in the sweet-substance anal- 296 Pharmacological Reports, 2012, 64,

5 Antinociceptive activity of aspartame and sucrose Seema Rani and Mahesh C. Gupta gesia [11]. In fact, there is also evidence for the involvement of the locus coeruleus neurons in at least sucrose-induced antinociception as previously described [12, 16]. Thus, the various neurochemical mechanisms of pain can give us new ways to understand central pain inhibitory pathways. Endogenous opioids and serotonin may be involved in the central regulation of the sweet substance-produced analgesia in adult rodents. Further studies are needed to find out the possible therapeutic place of sucrose and aspartame in pain management in human. In conclusion, aspartame showed an antinociceptive activity similar to sucrose. Nociceptive activity of both aspartame and sucrose was reduced after peripheral treatment with opioid and serotoninergic antagonists, thereby showing involvement of opioid system and serotonergic system in analgesia induced by these artificial sweeteners. References: 1. Abdollahi M, Nikfar S, Aghabarati F, Etemad F, Abdoli N: Interaction of different doses of aspartame with morphine-induced antinociception in presence of MK-801, a NMDA antagonist. DARU, 2002, 10, Anseloni VC, Weng HR, Terayama R, Letizia D, Davis BJ, Ren K, Dubner R et al.: Age-dependency of analgesia elicited by intraoral sucrose in acute and persistent pain models. Pain, 2002, 97, Bergmann F, Cohen E, Lieblich I: Biphasic effects of chronic saccharin intake on pain responses of healthy and diabetic rats of two genetically selected strains. Psychopharmacology, 1984, 82, Bergmann F, Lieblich I, Cohen E, Ganchrow JR: Influence of intake of sweet solutions on the analgesic effect of a low dose of morphine in randomly bred rats. Behav Neural Biol, 1985, 44, Blass EM, Fitzgerald E: Milk-induced analgesia and comforting in 10-day-old rats: opioid mediation. Pharmacol Biochem Behav, 1988, 29, Blass EM, Shide DJ: Some comparisons among the calming and pain-relieving effects of sucrose, glucose, fructose and lactose in infant rats. Chem Senses, 1994, 19, D Anci KE, Kanarek RB, Marks-Kaufman R: Beyond sweet taste: saccharin, sucrose, and polycose differ in their effects upon morphine-induced analgesia. Pharmacol Biochem Behav, 1997, 56, Haouari N, Wood C, Griffiths G, Levene M: The analgesic effect of sucrose in full term infants: a randomised controlled trial. BMJ, 1995, 310, Holder MD: Responsivity to pain in rats changed by the ingestion of flavoured water. Behav Neural Biol, 1988, 49, Irusta AEC, Savoldi M, Kishi R, Resende GCC, Freitas RL, Carvalho AD, Coimbra NC: Psychopharmacological evidences for the involvement of muscarinic and nicotinic cholinergic receptors on sweet substance-induced analgesia in Rattus norvegicus. Neurosci Lett, 2001, 305, Israilova M, Suzuki F, Tanaka T, Nagatomo T, Taniguchi T, Muramatsu I: Binding and functional affinity of sarpogrelate, its metabolite m-1 and ketanserin for human recombinant alpha-1-adrenoceptor subtypes. Pharmacology, 2002, 65, Kishi R, Bongiovanni R, de Nadai TR, Freitas RL, de Oliveira R, Ferreira CM, Coimbra NC: Dorsal raphe nucleus and locus coeruleus neural networks and the elaboration of the sweet-substance-induced antinociception. Neurosci Lett, 2006, 395, Lieblich I, Cohen E, Ganchrow JR, Blass EM, Bergmann F: Morphine tolerance in genetically selected rats induced by chronically elevated saccharine intake. Science, 1983, 221, Mayer DJ, Liebeskind JC: Pain reduction by focal electrical stimulation of the brain: an anatomical and behavioural analysis. Brain Res, 1974, 68, McNally GP, Westbrook RF: Acute exposure to saccharin reduces morphine analgesia in the rats: evidence of involvement of N-methyl-D-aspartate and peripheral opioid receptors. Psychopharmacology, 2000, 149, Miyase CI, Kishi R, de Freitas RL, Paz DA, Coimbra NC.: Involvement of pre- and post-synaptic serotonergic receptors of dorsal raphe nucleus neural network in the control of the sweet-substance-induced analgesia in adult Rattus norvegicus (Rodentia, Muridae). Neurosci Lett, 2005, 379, Nikfar S, Abdollahi M, Etemad F, Sharifzadeh M: Effects of sweetening agents on morphine-induced antinociception in mice by formalin-test. Gen Pharmacol, 1997, 29, Rebouças ECC, Segato E, Kishi R Freitas RL, Savoldi M, Morato S, Coimbra NC: Effect of the blockade of µ 1 -opioid and 5HT 2A serotonergic/ 1-noradrenergic receptors on sweet-substance-induced analgesia. Psychopharmacology, 2005, 179, Roane DS, Martin RJ: Continuous sucrose feeding decreases pain threshold and increases morphine potency. Pharmacol Biochem Behav, 1990, 35, Segato FN, Castro-Souza C, Segato EM, Morato S, Coimbra NC: Sucrose ingestion causes opioid analgesia. Braz J Med Biol Res, 1997, 30, Segato EN, Reboucas ECC, Freitas RL, Caires MPT, Cardoso AV, Resende GCC, Shimizu-Bassi G et al.: Effect of chronic intake of sweet substance on nociceptive thresholds and feeding behavior of Rattus norvegicus. Nutr Neurosci, 2005, 8, Suh HW, Song DK, Wie MB, Jung JS, Hong HE, Choi SR, Kim YH: The reduction of antinociceptive effect of morphine administered intraventricularly is correlated with the decrease of serotonin release from the spinal Pharmacological Reports, 2012, 64,

6 cord in streptozotocin-induced diabetic rats. Gen Pharmacol, 1996, 27, Takeshita N, Ohkubo Y, Yamaguchi I: Tiapride attenuates pain transmission through an indirect activation of central serotonergic mechanism. J Pharmacol Exp Ther, 1995, 275, Takeshita N, Yamaguchi I: Meta-chlorophenylpiperazine attenuates formalin-induced nociceptive responses through 5-HT 1/2 receptors in both normal and diabetic mice. Br J Pharmacol, 1995, 116, Yaksh TL, Rudy TA: Analgesia mediated by a direct spinal action of narcotics. Science, 1976, 192, Received: April 1, 2011; in the revised form: December 7, 2011; accepted: December 13, Pharmacological Reports, 2012, 64,