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Involvement of serotonergic system in the antidepressant-like effect of piperine
Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 1144–1147
Contents lists available at ScienceDirect
Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p
Involvement of serotonergic system in the antidepressant-like effect of piperine Qing-Qiu Mao, Yan-Fang Xian, Siu-Po Ip ⁎, Chun-Tao Che School of Chinese Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong
a r t i c l e
i n f o
Article history: Received 13 December 2010 Received in revised form 28 March 2011 Accepted 28 March 2011 Available online 6 April 2011 Keywords: Antidepressant Mice Piperine Serotonergic system
a b s t r a c t Piperine is a major alkaloid of black pepper (Piper nigrum Linn.) and long pepper (P. longum Linn.), and its antidepressant-like effect has been previously demonstrated. The purpose of this study was to explore the possible contribution of the serotonergic system in the antidepressant-like effect of piperine in mice. The results showed that piperine significantly reduced the immobility time in the forced swim test and tail suspension test in mice. The anti-immobility effect of piperine in the forced swim test and tail suspension test was completely abolished by pre-treating the mice with pCPA (an inhibitor of 5-HT synthesis). Piperine treatment also significantly potentiated the number of head-twitches of mice induced by 5-HTP (a metabolic precursor to 5-HT). In addition, the neurochemical assays showed that piperine produced a marked increase of 5-HT level in both the hippocampus and frontal cortex of mice. Taken together, these results clearly suggest that the antidepressant-like effect of piperine is mediated via the serotonergic system by enhancing 5-HT content in mouse brain. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Depression, a common psychiatric disorder characterized by a pervasive low mood, loss of interest or pleasure in daily activities, affects up to 21% of the world's population (McKenna et al., 2005). The World Health Organization estimates that depression will be the second most prevalent cause of loss in human disability adjusted life years worldwide by the year 2020 (Murray and Lopez, 1996). Many studies of depression have paid special attention to the serotonergic system. The decrease of brain serotonin (5-HT) activity was commonly observed in animals and in patients experiencing stress and depression (Risch and Nemeroff, 1992; Spreux-Varoquaux et al., 2001; An et al., 2008), while drugs that are acted by increasing the bioavailability of brain 5-HT, such as selective 5-HT reuptake inhibitors and monoamine oxidase inhibitors, are widely used in clinical depression treatment (Millan, 2004; Krishnan and Nestler, 2008). Furthermore, 5-HT and its receptors have been found to be involved in the mechanism of action of antidepressants in different behavioral studies (Einat et al., 2001; An et al., 2008; Szewczyk et al., 2009; Zhu et al., 2009; Freitas et al., 2010; Yi et al., 2010). These data suggest that brain serotonergic system plays an important role in the pathogenesis and therapy of depression (Krishnan and Nestler, 2008). Piperine, a major alkaloid of black pepper (Piper nigrum Linn.) and long pepper (P. longum Linn.), has been used extensively as condiment and flavoring for all types of savory dishes (Li et al., Abbreviations: 5-HT, serotonin; 5-HTP, 5-hydroxy-L-tryptophan; i.p., intraperitoneally; pCPA, p-chlorophenylalanine. ⁎ Corresponding author. Tel.: + 852 3163 4457; fax: + 852 3163 4459. E-mail address: [email protected] (S.-P. Ip). 0278-5846/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2011.03.017
2007a,b). In recent years, pharmacological studies have showed that piperine possesses analgesic, anti-inflammatory, anti-convulsant, anti-oxidant and cognitive enhancing effects (Gupta et al., 2000; Selvendiran et al., 2003; Chonpathompikunlert et al., 2010). Piperine has been reported to inhibit monoamine oxidase activity, increase monoamine neurotransmitter levels, and thus produce antidepressant-like activity in various mouse models of behavioral despair (Lee et al., 2005; Li et al., 2007a). The antidepressive effect of piperine has also been observed in mice exposed to chronic mild stress, which were related to up-regulation of hippocampal progenitor cell proliferation (Li et al., 2007b). However, there has been only sparse information about the interaction between serotonergic system and the antidepressant-like effect of piperine. Therefore, the present study was designed to evaluate the involvement of brain serotonergic system in the antidepressant-like effect of piperine in mice, by using behavioral and neurochemical assays. 2. Materials and methods 2.1. Animals Male ICR mice weighing 18–25 g were obtained from the Laboratory Animal Services Center, The Chinese University of Hong Kong, Hong Kong. The animals were maintained on a 12-hour light/dark cycle under regulated temperature (22 ± 2 °C) and humidity (50 ± 10 %) and fed with standard diet and water ad libitum. They were allowed to acclimate 3 days before use. The experiments on animals have been approved by Department of Health, The Government of the Hong Kong Special Administrative Region and conformed to the guidelines of the “Principles of Laboratory Animal Care” (NIH publication No.80-23,
Q.-Q. Mao et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 1144–1147
Piperine, fluoxetine, 5-hydroxy-L-tryptophan (5-HTP) and pchlorophenylalanine (pCPA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All drugs were dissolved in saline, except piperine was dissolved in saline after being dispersed with Tween-80 and the final concentration of Tween-80 is less than 0.1%. Drugs and vehicle were all administered intraperitoneally (i.p.) in a volume of 10 ml/kg. Observable adverse effect such as writhing behavior was not seen following the injection. 2.3. The depletion of 5-HT in the forced swim test and tail suspension test Mice were pre-treated with pCPA (300 mg/kg) or saline once per day for 3 consecutive days (An et al., 2008). 24 h after the last pCPA administration, mice were administered with saline, piperine (5 and 10 mg/kg) or fluoxetine (10 mg/kg). Forty-five minutes later, the animals were investigated by forced swim test and tail suspension test. The forced swim test was carried out according to the method described by Porsolt et al. (1977). Briefly, mice were forced to swim in a transparent glass vessel (25 cm high, 14 cm in diameter) filled with 10 cm of water at 24–26 °C. The total duration of immobility (seconds) was measured during the last 4 min of a single 6-min test session. Mice were considered immobile when they made no attempts to escape except the movements necessary to keep their heads above the water. The tail suspension test was carried out according to the method of Steru et al. (1985). Briefly, mice were suspended 5 cm above the floor by means of an adhesive tape, placed approximately 1 cm from the tip of the tail. The total duration of immobility (seconds) was quantified during a test period of 6 min. Mice were considered immobile when they were completely motionless. 2.4. 5-HTP-induced head-twitches test 5-HTP-induced head-twitches test was carried out as described previously (An et al., 2008). Briefly, mice were administered with saline, piperine (5 and 10 mg/kg) or fluoxetine (10 mg/kg) 45 min before injection of 5-HTP (120 mg/kg). Immediately after the injection, mice were placed into plastic cages and the cumulative number of head-twitches (rapid movements of the head with little or no involvement of the trunk) was recorded during a test period of 20 min. 2.5. Measurement of 5-HT content Mice were administered with saline, piperine (5 and 10 mg/kg) or fluoxetine (10 mg/kg). Forty-five minutes after administration, mice were sacrificed by decapitation. Whole brains were rapidly removed from mice and chilled in an ice-cold saline. Various brain areas, including hippocampus and frontal cortex, were dissected on a cold plate and frozen in liquid nitrogen immediately. The tissue samples were stored at −80 °C until assay. Hippocampus and frontal cortex samples were homogenized in 10 volumes of perchloric acid (0.2 N), and centrifuged at 10,000 g for 5 min. The supernatant was removed, neutralized with equal volume of 1.0 M borate buffer (pH 9.25) and centrifuged at 10,000 g for 1 min. The resulting supernatant was used for 5-HT assay by using an immunoassay kit (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions. The absorbance was measured at 405 nm. The 5-HT level was expressed as ng/g wet weight of tissue.
Data were expressed as mean ± SEM. The effects of piperine when compared to the control group were established by one-way analysis of variance (ANOVA) followed by Dunnett test. In the interaction studies, statistical analysis was performed using two-way ANOVA followed by Bonferroni test. The GraphPad Prism software was used to perform the statistics (version 4.0; GraphPad Software, Inc., San Diego, CA). The difference was considered statistically significant when p b 0.05. 3. Results One-way ANOVA showed a significant effect of drug treatment on the duration of immobility in the forced swim test (F (3, 28) = 5.44, p b 0.01). Post-hoc analysis indicated a significant decrease in the immobility time in the forced swim test elicited by piperine at the doses of 5, 10 mg/kg and fluoxetine at 10 mg/kg, when compared to the control group (Fig. 1A). One-way ANOVA also showed a significant effect of drug treatment on the duration of immobility in the tail suspension test (F (3, 36) = 9.18, p b 0.01). Post-hoc analysis indicated a significant decrease in the immobility time in the tail suspension test elicited by piperine at the doses of 5, 10 mg/kg and fluoxetine at 10 mg/kg, when compared to the control group (Fig. 1B). Fig. 1 also showed the effect of pre-treatment with pCPA on the reduction of immobility time elicited by piperine and fluoxetine in the forced swim test and tail suspension test. The two-way ANOVA revealed a significant difference of treatment (piperine or fluoxetine; F(3, 56) = 26.11, p b 0.01) without effect of pre-treatment (saline or pCPA; F(1, 56) = 2.59, p = 0.06) and pre-treatment × treatment interaction (F(3, 56) = 2.54, p = 0.06) on the immobility time in the forced swim test. However, although not statistically significant, there is a tendency of pre-treatment of mice with pCPA to block the
A Duration of immobility (s)
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2.6. Statistical analysis
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revised 1996). All efforts were made to minimize animals suffering and to reduce the number of animals used in the experiments.
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Fig. 1. Effect of pre-treatment with pCPA on the anti-immobility effect of piperine in the forced swim test (A) and tail suspension test (B) in mice. Mice were pre-treated with pCPA (300 mg/kg, i.p.) or saline for 3 consecutive days. 24 h after the last pCPA administration, piperine and fluoxetine were administered intraperitoneally 45 min before the test. Data were expressed as the mean ± SEMs of 8–10 animals. * p b 0.05, ** p b 0.01 as compared with the control group, #p b 0.01 as compared with the same group pre-treated with saline.
Q.-Q. Mao et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 1144–1147
A 800 5-HT level (ng/g tissue)
decrease in immobility in the forced swim test elicited by piperine or fluoxetine. Moreover, the two-way ANOVA revealed significant differences of pre-treatment (saline or pCPA; F(1, 72) = 4.53, p b 0.01), treatment (F(3, 72) = 39.76, p b 0.01), and pre-treatment × treatment interaction (F(3, 72) = 6.20, p b 0.01) on the immobility time in the tail suspension test. Post hoc analysis indicated that the pre-treatment of mice with pCPA significantly blocked the decrease in immobility in the tail suspension test elicited by piperine or fluoxetine. pCPA alone did not modify the immobility time in the forced swim test and tail suspension test. As shown in Fig. 2, one-way ANOVA showed a significant effect of drug treatment on the cumulative number of head-twitches of mice induced by 5-HTP (F (3, 36) = 25.61, p b 0.01). Post-hoc analysis indicated a significant increase in the cumulative number of headtwitches of mice induced by 5-HTP elicited by piperine at the doses of 5, 10 mg/kg and fluoxetine at 10 mg/kg, when compared to the control group. As shown in Fig. 3A, one-way ANOVA showed a significant effect of drug treatment on 5-HT level in the hippocampus of mice (F (3, 20) = 6.36, p b 0.01). Post-hoc analysis indicated a significant increase in 5HT level in the hippocampus of mice elicited by the administration of piperine at the doses of 5, 10 mg/kg and fluoxetine at 10 mg/kg, when compared to the control group. One-way ANOVA also showed a significant effect of drug treatment on 5-HT level in the frontal cortex of mice (F (3, 20) = 4.86, p b 0.05). Post-hoc analysis indicated a significant increase in 5-HT level in the frontal cortex of mice elicited by piperine at the dose of 10 mg/kg and fluoxetine at 10 mg/kg, when compared to the control group.
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Fig. 3. Effect of piperine treatment on 5-HT level in the hippocampus (A) and frontal cortex (B) of mice. Piperine and fluoxetine were administered intraperitoneally 45 min before the assays. Data were expressed as the mean ± SEMs of 6 animals. * p b 0.05 and ** p b 0.01 as compared with the control group.
4. Discussion
Number of head twitches
The forced swim test and tail suspension test are the widely used behavioral tests for the screening of antidepressants, largely due to its strong predictive validity and its ability to detect activity of a broad spectrum of clinically effective antidepressants (Porsolt et al., 1977; Steru et al., 1985, 1987). In these tests, animals are under stress from which they cannot escape. After an initial period of struggling, they would become immobile, resembling a state of despair and mental depression (Porsolt et al., 1977; Steru et al., 1985, 1987). It has been reported that treating mice intragastrically with piperine for two weeks significantly reduced the duration of immobility in the forced swim test and tail suspension test, without producing locomotor alteration in the open-field test (Li et al., 2007a). Consistently, in the present study, single piperine treatment (5 and 10 mg/kg, i.p.) was also found to produce antidepressive action in the forced swim test and tail suspension test in mice. The present study aims to prove the possible contribution of the serotonergic system to the antidepressant-like effect of piperine. The
25 **
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Fig. 2. Effect of piperine treatment on the number of head twitches of mice induced by 5-HTP. Piperine and fluoxetine were administered intraperitoneally 45 min before the test. Data were expressed as the mean ± SEMs of 10 animals. ** p b 0.01 as compared with the control group.
effect of pCPA, an inhibitor of 5-HT synthesis, on the antidepressantlike effect of piperine was measured by the forced swim test and tail suspension test. Previous study has showed that treating the animals with pCPA 150 mg/kg cause a depletion of cortical 5-HT content by 93% (Page et al., 1999). Moreover, pCPA treatment could completely abrogate the antidepressant-like effect of fluoxetine, a selective 5-HT reuptake inhibitor, while it only partially reversed the effect of imipramine, an inhibitor of 5-HT and noradrenaline (Eckeli et al., 2000). In line with previous findings (Eckeli et al., 2000; An et al., 2008), the results presented here showed that pCPA alone did not modify the immobility time, while pre-treating the mice with pCPA significantly blocked the reduction in the immobility time elicited by piperine and fluoxetine in the forced swim test and tail suspension test. The ability of pCPA to eliminate the antidepressant-like effect of piperine supported the hypothesis that the behavioral effects of piperine require the serotonergic system to be intact. 5-HTP-induced head-twitches test is usually used to investigate both acute and long-term effects of antidepressive drugs on serotonergic function (An et al., 2008). The present study revealed that piperine treatment significantly increased the number of headtwitches of mice induced by 5-HTP, reinforcing the assumption that serotonergic system is involved in the antidepressant-like effect of piperine. To support the hypothesis that the antidepressant-like effect of piperine is mediated by the increase of 5-HT content, the effect of piperine on 5-HT content in mouse brain was studied. In the present study, we focused on two brain regions, the hippocampus and the frontal cortex. These two brain regions are both critically involved in the regulation of emotion, motivation, learning and memory, all of which may be related to the expression of depression (Qi et al., 2008). Our results showed that piperine administration produced a marked increase of 5-HT level in both hippocampus and frontal cortex, suggesting that the antidepressant-like effect of piperine might be related to the increase of 5-HT content in the mouse brain.
Q.-Q. Mao et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 1144–1147
5. Conclusion In conclusion, the present data indicate that the antidepressantlike effect of piperine is mediated via the serotonergic system by enhancing 5-HT content in mouse brain. Moreover, further studies will be conducted to examine the interaction between 5-HT receptors and the antidepressant-like effect of piperine. References An L, Zhang YZ, Yu NJ, Liu XM, Zhao N, Yuan L, et al. Role for serotonin in the antidepressant-like effect of a flavonoid extract of Xiaobuxin-Tang. Pharmacol Biochem Behav 2008;89:572–80. Chonpathompikunlert P, Wattanathorn J, Muchimapura S. Piperine, the main alkaloid of Thai black pepper, protects against neurodegeneration and cognitive impairment in animal model of cognitive deficit like condition of Alzheimer's disease. Food Chem Toxicol 2010;48:798–802. Eckeli AL, Dach F, Rodrigues AL. Acute treatment with GMP produce antidepressant-like effects in mice. Neuroreport 2000;11:1839–43. Einat H, Clenet F, Shaldubina A, Belmaker RH, Bourin M. The antidepressant activity of inositol in the forced swim test involves 5-HT(2) receptors. Behav Brain Res 2001;118:77–83. Freitas AE, Budni J, Lobato KR, Binfaré RW, Machado DG, Jacinto J, et al. Antidepressantlike action of the ethanolic extract from Tabebuia avellanedae in mice: evidence for the involvement of the monoaminergic system. Prog Neuropsychopharmacol Biol Psychiatry 2010;34:335–43. Gupta SK, Bansal P, Bhardwaj RK, Velpandian T. Comparative antinociceptive, antiinflammatory and toxicity profile of nimesulide vs nimesulide and piperine combination. Pharmacol Res 2000;41:657–62. Lee SA, Hong SS, Han XH, Hwang JS, Oh GJ, Lee KS, et al. Piperine from the fruits of Piper longum with inhibitory effect on monoamine oxidase and antidepressant-like activity. Chem Pharm Bull 2005;53:832–5. Li S, Wang C, Li W, Koike K, Nikaido T, Wang MW. Antidepressant-like effects of piperine and its derivative, antiepilepsirine. J Asian Nat Prod Res 2007a;9:421–30. Li S, Wang C, Wang M, Li W, Matsumoto K, Tang Y. Antidepressant like effects of piperine in chronic mild stress treated mice and its possible mechanisms. Life Sci 2007b;15:1373–81.
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Krishnan V, Nestler EJ. The molecular neurobiology of depression. Nature 2008;455: 894–902. McKenna MT, Michaud CM, Murray CJL, Marks JS. Assessing the burden of disease in the United States using disability-adjusted life years. Am J Prev Med 2005;28:415–23. Millan MJ. The role of monoamines in the actions of established and “novel” antidepressant agents: a critical review. Eur J Pharmacol 2004;500:371–84. Murray CJ, Lopez AD. The global burden of disease: a comprehensive assessment of mortality and disability from diseases, injuries, and risk factors in 1990 and projected to 2020. Cambridge, Mass: Harvard University Press; 1996. Page ME, Detke MJ, Dalvi A, Kirby LG, Lucki I. Serotonergic mediation of the effects of fluoxetine, but not desipramine, in the rat forced swimming test. Psychopharmacology 1999;147:162–7. Porsolt RD, Pichon MLE, Jalfre M. Behavioral despair in mice: a primary screening test for antidepressant. Arch Int Pharmacodyn Ther 1977;229:327–36. Qi X, Lin W, Li J, Li H, Wang W, Wang D, et al. Fluoxetine increases the activity of the ERK-CREB signal system and alleviates the depressive-like behavior in rats exposed to chronic forced swim stress. Neurobiol Dis 2008;31:278–85. Risch SC, Nemeroff CB. Neurochemical alterations of serotonergic neuronal systems in depression. J Clin Psychiatry 1992;53:3–7. Selvendiran K, Singh JP, Krishnan KB, Sakthisekaran D. Cytoprotective effect of piperine against benzo[a]pyrene induced lung cancer with reference to lipid peroxidation and antioxidant system in Swiss albino mice. Fitoterapia 2003;74:109–15. Spreux-Varoquaux O, Alvarez JC, Berlin I, Batista G, Despierre PG, Gilton A, et al. Differential abnormalities in plasma 5-HIAA and platelet serotonin concentrations in violent suicide attempters: relationships with impulsivity and depression. Life Sci 2001;69:647–57. Steru L, Chermat R, Thierry B, Simon P. The tail suspension test: a new method for screening antidepressants in mice. Psychopharmacology 1985;85:367–70. Steru L, Chermat R, Thierry B, Mico JA, Lenegre A, Steru M, et al. The automated tail suspension test: a computerized device which differentiates psychotropic drugs. Prog Neuropsychopharmacol Biol Psychiatry 1987;11:659–71. Szewczyk B, Poleszak E, Wlaź P, Wróbel A, Blicharska E, Cichy A, et al. The involvement of serotonergic system in the antidepressant effect of zinc in the forced swim test. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:323–9. Yi LT, Li CF, Zhan X, Cui CC, Xiao F, Zhou LP, et al. Involvement of monoaminergic system in the antidepressant-like effect of the flavonoid naringenin in mice. Prog Neuropsychopharmacol Biol Psychiatry 2010;34:1223–8. Zhu H, Huang Q, Xu H, Niu L, Zhou JN. Antidepressant-like effects of sodium butyrate in combination with estrogen in rat forced swimming test: involvement of 5-HT(1A) receptors. Behav Brain Res 2009;196:200–6.
Contents lists available at ScienceDirect
Progress in Neuro-Psychopharmacology & Biological Psychiatry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p n p
Involvement of serotonergic system in the antidepressant-like effect of piperine Qing-Qiu Mao, Yan-Fang Xian, Siu-Po Ip ⁎, Chun-Tao Che School of Chinese Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong
a r t i c l e
i n f o
Article history: Received 13 December 2010 Received in revised form 28 March 2011 Accepted 28 March 2011 Available online 6 April 2011 Keywords: Antidepressant Mice Piperine Serotonergic system
a b s t r a c t Piperine is a major alkaloid of black pepper (Piper nigrum Linn.) and long pepper (P. longum Linn.), and its antidepressant-like effect has been previously demonstrated. The purpose of this study was to explore the possible contribution of the serotonergic system in the antidepressant-like effect of piperine in mice. The results showed that piperine significantly reduced the immobility time in the forced swim test and tail suspension test in mice. The anti-immobility effect of piperine in the forced swim test and tail suspension test was completely abolished by pre-treating the mice with pCPA (an inhibitor of 5-HT synthesis). Piperine treatment also significantly potentiated the number of head-twitches of mice induced by 5-HTP (a metabolic precursor to 5-HT). In addition, the neurochemical assays showed that piperine produced a marked increase of 5-HT level in both the hippocampus and frontal cortex of mice. Taken together, these results clearly suggest that the antidepressant-like effect of piperine is mediated via the serotonergic system by enhancing 5-HT content in mouse brain. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Depression, a common psychiatric disorder characterized by a pervasive low mood, loss of interest or pleasure in daily activities, affects up to 21% of the world's population (McKenna et al., 2005). The World Health Organization estimates that depression will be the second most prevalent cause of loss in human disability adjusted life years worldwide by the year 2020 (Murray and Lopez, 1996). Many studies of depression have paid special attention to the serotonergic system. The decrease of brain serotonin (5-HT) activity was commonly observed in animals and in patients experiencing stress and depression (Risch and Nemeroff, 1992; Spreux-Varoquaux et al., 2001; An et al., 2008), while drugs that are acted by increasing the bioavailability of brain 5-HT, such as selective 5-HT reuptake inhibitors and monoamine oxidase inhibitors, are widely used in clinical depression treatment (Millan, 2004; Krishnan and Nestler, 2008). Furthermore, 5-HT and its receptors have been found to be involved in the mechanism of action of antidepressants in different behavioral studies (Einat et al., 2001; An et al., 2008; Szewczyk et al., 2009; Zhu et al., 2009; Freitas et al., 2010; Yi et al., 2010). These data suggest that brain serotonergic system plays an important role in the pathogenesis and therapy of depression (Krishnan and Nestler, 2008). Piperine, a major alkaloid of black pepper (Piper nigrum Linn.) and long pepper (P. longum Linn.), has been used extensively as condiment and flavoring for all types of savory dishes (Li et al., Abbreviations: 5-HT, serotonin; 5-HTP, 5-hydroxy-L-tryptophan; i.p., intraperitoneally; pCPA, p-chlorophenylalanine. ⁎ Corresponding author. Tel.: + 852 3163 4457; fax: + 852 3163 4459. E-mail address: [email protected] (S.-P. Ip). 0278-5846/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2011.03.017
2007a,b). In recent years, pharmacological studies have showed that piperine possesses analgesic, anti-inflammatory, anti-convulsant, anti-oxidant and cognitive enhancing effects (Gupta et al., 2000; Selvendiran et al., 2003; Chonpathompikunlert et al., 2010). Piperine has been reported to inhibit monoamine oxidase activity, increase monoamine neurotransmitter levels, and thus produce antidepressant-like activity in various mouse models of behavioral despair (Lee et al., 2005; Li et al., 2007a). The antidepressive effect of piperine has also been observed in mice exposed to chronic mild stress, which were related to up-regulation of hippocampal progenitor cell proliferation (Li et al., 2007b). However, there has been only sparse information about the interaction between serotonergic system and the antidepressant-like effect of piperine. Therefore, the present study was designed to evaluate the involvement of brain serotonergic system in the antidepressant-like effect of piperine in mice, by using behavioral and neurochemical assays. 2. Materials and methods 2.1. Animals Male ICR mice weighing 18–25 g were obtained from the Laboratory Animal Services Center, The Chinese University of Hong Kong, Hong Kong. The animals were maintained on a 12-hour light/dark cycle under regulated temperature (22 ± 2 °C) and humidity (50 ± 10 %) and fed with standard diet and water ad libitum. They were allowed to acclimate 3 days before use. The experiments on animals have been approved by Department of Health, The Government of the Hong Kong Special Administrative Region and conformed to the guidelines of the “Principles of Laboratory Animal Care” (NIH publication No.80-23,
Q.-Q. Mao et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 1144–1147
Piperine, fluoxetine, 5-hydroxy-L-tryptophan (5-HTP) and pchlorophenylalanine (pCPA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All drugs were dissolved in saline, except piperine was dissolved in saline after being dispersed with Tween-80 and the final concentration of Tween-80 is less than 0.1%. Drugs and vehicle were all administered intraperitoneally (i.p.) in a volume of 10 ml/kg. Observable adverse effect such as writhing behavior was not seen following the injection. 2.3. The depletion of 5-HT in the forced swim test and tail suspension test Mice were pre-treated with pCPA (300 mg/kg) or saline once per day for 3 consecutive days (An et al., 2008). 24 h after the last pCPA administration, mice were administered with saline, piperine (5 and 10 mg/kg) or fluoxetine (10 mg/kg). Forty-five minutes later, the animals were investigated by forced swim test and tail suspension test. The forced swim test was carried out according to the method described by Porsolt et al. (1977). Briefly, mice were forced to swim in a transparent glass vessel (25 cm high, 14 cm in diameter) filled with 10 cm of water at 24–26 °C. The total duration of immobility (seconds) was measured during the last 4 min of a single 6-min test session. Mice were considered immobile when they made no attempts to escape except the movements necessary to keep their heads above the water. The tail suspension test was carried out according to the method of Steru et al. (1985). Briefly, mice were suspended 5 cm above the floor by means of an adhesive tape, placed approximately 1 cm from the tip of the tail. The total duration of immobility (seconds) was quantified during a test period of 6 min. Mice were considered immobile when they were completely motionless. 2.4. 5-HTP-induced head-twitches test 5-HTP-induced head-twitches test was carried out as described previously (An et al., 2008). Briefly, mice were administered with saline, piperine (5 and 10 mg/kg) or fluoxetine (10 mg/kg) 45 min before injection of 5-HTP (120 mg/kg). Immediately after the injection, mice were placed into plastic cages and the cumulative number of head-twitches (rapid movements of the head with little or no involvement of the trunk) was recorded during a test period of 20 min. 2.5. Measurement of 5-HT content Mice were administered with saline, piperine (5 and 10 mg/kg) or fluoxetine (10 mg/kg). Forty-five minutes after administration, mice were sacrificed by decapitation. Whole brains were rapidly removed from mice and chilled in an ice-cold saline. Various brain areas, including hippocampus and frontal cortex, were dissected on a cold plate and frozen in liquid nitrogen immediately. The tissue samples were stored at −80 °C until assay. Hippocampus and frontal cortex samples were homogenized in 10 volumes of perchloric acid (0.2 N), and centrifuged at 10,000 g for 5 min. The supernatant was removed, neutralized with equal volume of 1.0 M borate buffer (pH 9.25) and centrifuged at 10,000 g for 1 min. The resulting supernatant was used for 5-HT assay by using an immunoassay kit (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions. The absorbance was measured at 405 nm. The 5-HT level was expressed as ng/g wet weight of tissue.
Data were expressed as mean ± SEM. The effects of piperine when compared to the control group were established by one-way analysis of variance (ANOVA) followed by Dunnett test. In the interaction studies, statistical analysis was performed using two-way ANOVA followed by Bonferroni test. The GraphPad Prism software was used to perform the statistics (version 4.0; GraphPad Software, Inc., San Diego, CA). The difference was considered statistically significant when p b 0.05. 3. Results One-way ANOVA showed a significant effect of drug treatment on the duration of immobility in the forced swim test (F (3, 28) = 5.44, p b 0.01). Post-hoc analysis indicated a significant decrease in the immobility time in the forced swim test elicited by piperine at the doses of 5, 10 mg/kg and fluoxetine at 10 mg/kg, when compared to the control group (Fig. 1A). One-way ANOVA also showed a significant effect of drug treatment on the duration of immobility in the tail suspension test (F (3, 36) = 9.18, p b 0.01). Post-hoc analysis indicated a significant decrease in the immobility time in the tail suspension test elicited by piperine at the doses of 5, 10 mg/kg and fluoxetine at 10 mg/kg, when compared to the control group (Fig. 1B). Fig. 1 also showed the effect of pre-treatment with pCPA on the reduction of immobility time elicited by piperine and fluoxetine in the forced swim test and tail suspension test. The two-way ANOVA revealed a significant difference of treatment (piperine or fluoxetine; F(3, 56) = 26.11, p b 0.01) without effect of pre-treatment (saline or pCPA; F(1, 56) = 2.59, p = 0.06) and pre-treatment × treatment interaction (F(3, 56) = 2.54, p = 0.06) on the immobility time in the forced swim test. However, although not statistically significant, there is a tendency of pre-treatment of mice with pCPA to block the
A Duration of immobility (s)
2.2. Drugs
2.6. Statistical analysis
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revised 1996). All efforts were made to minimize animals suffering and to reduce the number of animals used in the experiments.
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Fig. 1. Effect of pre-treatment with pCPA on the anti-immobility effect of piperine in the forced swim test (A) and tail suspension test (B) in mice. Mice were pre-treated with pCPA (300 mg/kg, i.p.) or saline for 3 consecutive days. 24 h after the last pCPA administration, piperine and fluoxetine were administered intraperitoneally 45 min before the test. Data were expressed as the mean ± SEMs of 8–10 animals. * p b 0.05, ** p b 0.01 as compared with the control group, #p b 0.01 as compared with the same group pre-treated with saline.
Q.-Q. Mao et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 1144–1147
A 800 5-HT level (ng/g tissue)
decrease in immobility in the forced swim test elicited by piperine or fluoxetine. Moreover, the two-way ANOVA revealed significant differences of pre-treatment (saline or pCPA; F(1, 72) = 4.53, p b 0.01), treatment (F(3, 72) = 39.76, p b 0.01), and pre-treatment × treatment interaction (F(3, 72) = 6.20, p b 0.01) on the immobility time in the tail suspension test. Post hoc analysis indicated that the pre-treatment of mice with pCPA significantly blocked the decrease in immobility in the tail suspension test elicited by piperine or fluoxetine. pCPA alone did not modify the immobility time in the forced swim test and tail suspension test. As shown in Fig. 2, one-way ANOVA showed a significant effect of drug treatment on the cumulative number of head-twitches of mice induced by 5-HTP (F (3, 36) = 25.61, p b 0.01). Post-hoc analysis indicated a significant increase in the cumulative number of headtwitches of mice induced by 5-HTP elicited by piperine at the doses of 5, 10 mg/kg and fluoxetine at 10 mg/kg, when compared to the control group. As shown in Fig. 3A, one-way ANOVA showed a significant effect of drug treatment on 5-HT level in the hippocampus of mice (F (3, 20) = 6.36, p b 0.01). Post-hoc analysis indicated a significant increase in 5HT level in the hippocampus of mice elicited by the administration of piperine at the doses of 5, 10 mg/kg and fluoxetine at 10 mg/kg, when compared to the control group. One-way ANOVA also showed a significant effect of drug treatment on 5-HT level in the frontal cortex of mice (F (3, 20) = 4.86, p b 0.05). Post-hoc analysis indicated a significant increase in 5-HT level in the frontal cortex of mice elicited by piperine at the dose of 10 mg/kg and fluoxetine at 10 mg/kg, when compared to the control group.
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Fig. 3. Effect of piperine treatment on 5-HT level in the hippocampus (A) and frontal cortex (B) of mice. Piperine and fluoxetine were administered intraperitoneally 45 min before the assays. Data were expressed as the mean ± SEMs of 6 animals. * p b 0.05 and ** p b 0.01 as compared with the control group.
4. Discussion
Number of head twitches
The forced swim test and tail suspension test are the widely used behavioral tests for the screening of antidepressants, largely due to its strong predictive validity and its ability to detect activity of a broad spectrum of clinically effective antidepressants (Porsolt et al., 1977; Steru et al., 1985, 1987). In these tests, animals are under stress from which they cannot escape. After an initial period of struggling, they would become immobile, resembling a state of despair and mental depression (Porsolt et al., 1977; Steru et al., 1985, 1987). It has been reported that treating mice intragastrically with piperine for two weeks significantly reduced the duration of immobility in the forced swim test and tail suspension test, without producing locomotor alteration in the open-field test (Li et al., 2007a). Consistently, in the present study, single piperine treatment (5 and 10 mg/kg, i.p.) was also found to produce antidepressive action in the forced swim test and tail suspension test in mice. The present study aims to prove the possible contribution of the serotonergic system to the antidepressant-like effect of piperine. The
25 **
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Fig. 2. Effect of piperine treatment on the number of head twitches of mice induced by 5-HTP. Piperine and fluoxetine were administered intraperitoneally 45 min before the test. Data were expressed as the mean ± SEMs of 10 animals. ** p b 0.01 as compared with the control group.
effect of pCPA, an inhibitor of 5-HT synthesis, on the antidepressantlike effect of piperine was measured by the forced swim test and tail suspension test. Previous study has showed that treating the animals with pCPA 150 mg/kg cause a depletion of cortical 5-HT content by 93% (Page et al., 1999). Moreover, pCPA treatment could completely abrogate the antidepressant-like effect of fluoxetine, a selective 5-HT reuptake inhibitor, while it only partially reversed the effect of imipramine, an inhibitor of 5-HT and noradrenaline (Eckeli et al., 2000). In line with previous findings (Eckeli et al., 2000; An et al., 2008), the results presented here showed that pCPA alone did not modify the immobility time, while pre-treating the mice with pCPA significantly blocked the reduction in the immobility time elicited by piperine and fluoxetine in the forced swim test and tail suspension test. The ability of pCPA to eliminate the antidepressant-like effect of piperine supported the hypothesis that the behavioral effects of piperine require the serotonergic system to be intact. 5-HTP-induced head-twitches test is usually used to investigate both acute and long-term effects of antidepressive drugs on serotonergic function (An et al., 2008). The present study revealed that piperine treatment significantly increased the number of headtwitches of mice induced by 5-HTP, reinforcing the assumption that serotonergic system is involved in the antidepressant-like effect of piperine. To support the hypothesis that the antidepressant-like effect of piperine is mediated by the increase of 5-HT content, the effect of piperine on 5-HT content in mouse brain was studied. In the present study, we focused on two brain regions, the hippocampus and the frontal cortex. These two brain regions are both critically involved in the regulation of emotion, motivation, learning and memory, all of which may be related to the expression of depression (Qi et al., 2008). Our results showed that piperine administration produced a marked increase of 5-HT level in both hippocampus and frontal cortex, suggesting that the antidepressant-like effect of piperine might be related to the increase of 5-HT content in the mouse brain.
Q.-Q. Mao et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 35 (2011) 1144–1147
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