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Ferulic acid potentiates pentobarbital-induced sleep via the serotonergic system
Neuroscience Letters 525 (2012) 95–99
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Ferulic acid potentiates pentobarbital-induced sleep via the serotonergic system Yue Tu a,b , Shi-xiang Cheng a , Hong-tao Sun a , Tie-zhu Ma a , Sai Zhang a,∗ a b
Center for Neurology and Neurosurgery, Pingjin Hospital, Logistics College of Chinese People’s Armed Police Forces, Chenglin Road No. 220, Hedong District, Tianjin 300162, China Tianjin University of Traditional Chinese Medicine, Anshanxi Road No. 312, Nankai District, Tianjin 300193, China
h i g h l i g h t s
g r a p h i c a l
We investigated the sedative and hypnotic activities of ferulic acid. Ferulic acid inhibited the locomotion activity of mice. Ferulic acid potentiated the hypnotic effect of pentobarbital. Ferulic acid exerted sedative and hypnotic activity via the serotonergic system. Ferulic acid may be a pleasant candidate in therapy of insomnia.
Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a widespread natural phenolic compound which exert some bioactivity. The sedative and hypnotic effects of FA and possible mechanisms were investigated through behavioral pharmacology methods. The extract demonstrated that FA possessed sedative and hypnotic activities, which may mediated by serotonergic system.
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 20 March 2012 Received in revised form 23 July 2012 Accepted 29 July 2012 Keywords: Ferulic acid Sedative Hypnotic Pentobarbital Serotonergic system
a b s t r a c t
Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a widely distributed natural phenolic compound that is abundant in many plant tissues and foods. This study investigated possible mechanisms underlying the sedative–hypnotic effect of FA through behavioral pharmacology methods. FA showed dose-dependent sedative effects on locomotion activity in normal mice. FA also significantly potentiated pentobarbital-induced (45 mg/kg, i.p.) sleep by prolonging sleeping time and shortening sleep latency in a dose-dependent manner. These effects were augmented by the administration of 5hydroxytryptophan (5-HTP), a precursor of 5-hydroxytryptamine (5-HT). With a sub-hypnotic dose of pentobarbital (25 mg/kg, i.p.), FA significantly increased the rate of sleep onset and exhibited a synergistic effect with 5-HTP (2.5 mg/kg, i.p.). Pretreatment with p-chlorophenylalanine (PCPA, an inhibitor of tryptophan hydroxylase) significantly decreased the duration of pentobarbital-induced sleep, whereas FA significantly reversed this effect. These results suggest that FA has sedative–hypnotic activity, possibly mediated by the serotonergic system. © 2012 Elsevier Ireland Ltd. All rights reserved.
Insomnia, defined as persistent difficulty in initiating or maintaining sleep that affects daytime function, can induce significant psychological and physical disorders [10]. More than 27% of people worldwide experience insomnia, and approximately 3–10% of people are chronic and frequent users of hypnotics [7]. Clinically, benzodiazepines are the most widely used hypnotic agents
∗ Corresponding author. Tel.: +86 22 15216602576; fax: +86 22 60578627. E-mail address: [email protected] (S. Zhang). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.07.068
prescribed for insomnia. However, benzodiazepines have many unpleasant side effects, including drug dependence, drug tolerance, rebound insomnia, and amnesia. New types of hypnotics, such as zolpidem and zolpiclone, also show some side effects [2,9]. Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a ubiquitous natural phenolic acid found in many plant tissues and foods, including Ferula assafoetida L., Ligusticum chuanxiong Hort, Cimicifuga foetida L., cabbages, wheat, rice bran, tomatoes, and onions. FA exhibits many bioactivities including antioxidant, antimicrobial, radio-protective, hypertensive, and anti-carcinogenic properties
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[1,11,16]. Currently, FA is widely used in the medical, food, and cosmetic industries. Because FA has low toxicity and is metabolized quickly, it has been approved as a food additive in some countries. FA can pass through the blood-brain barrier and reach the brain; its pharmacokinetics complies with the open one-compartment model in vivo. It can be absorbed completely and rapidly, metabolized quickly, and excreted by the kidneys without apparent accumulation in vivo [4]. FA exerts neuro-protective effects against oxidative stress-related apoptosis after cerebral ischemia and significantly reduces cerebral infarct in a transient middle cerebral artery occlusion (MCAO) model [5,6,15]. Additional studies have reported that FA can attenuate neuronal cell death caused by the uptake of oxidized low-density lipoprotein, hydroxyl and peroxyl radicals in vitro [12,19]. FA may also attenuate neuronal cell death during MCAO involved in inhibition of Akt signaling pathway inactivation and maintenance of the interaction between phosphor-Bad and 14-3-3 [15]. FA may also have sedative activity. However, to the best of our knowledge, no previous studies have reported any hypnotic-sedative activity of FA. The present study investigated the hypnotic effects and possible mechanisms of FA on pentobarbitalinduced sleep in rodents. A total of 390 male ICR mice (weight 18–22 g, Grade I; purchased from Tianjin Medical University) were used in this study. The mice were housed under controlled environmental conditions (temperature 22 ± 2 ◦ C, humidity 50 ± 10%, 12 h light/dark cycle and light on at 0600) and given ad libitum access to food and water. The mice were acclimated 1 week before testing. The mice were fasted for 12 h prior to the onset of the experiments. The experiments were carried out between 0800 and 1300 in a quiet room with temperatures ranging from 22 to 24 ◦ C. All procedures were conducted in accordance with the European Community guidelines for the use of experimental animals and approved by the Tianjin Medical University Committee on Animal Care and Use. For intragastric (i.g.) administration (0.2 ml/10 g, volume/body weight), FA (Sigma–Aldrich, St. Louis) and l-malic acid (l-MA) were dissolved and diazepam injection (DZP, 10 mg/2 ml, manufactured by People’s Pharmaceutical Manufacturer, Tianjin, China) was diluted with 0.5% dimethyl sulfoxide (DMSO). For intraperitoneal (i.p.) injection (0.1 ml/10 g, volume/body weight), 5-HTP (Alfa Aesar China Ltd., Beijing, China) and pentobarbital (Serve, Shanghai Chemical Reagent Corporation, China) were dissolved with physiological saline. For subcutaneous (s.c.) injection (0.1 ml/10 g, volume/body weight), PCPA (Sigma–Aldrich, St. Louis) was suspended in 0.5% gum acacia/physiological saline. The locomotion activity of mice was measured using an YLS1A Multi-autonomous Activity Instrument with five activity cages (Shandong Academy of Medical Sciences, Jinan, China) [23]. FA and DZP were administered orally (0.2 ml/10 g body weight). Twentyfive minutes after injection, mice were acclimated to the activity cages individually for 5 min; then, the locomotion activity of each mouse was measured for 5 min. The present study used 45 mg/kg as the hypnotic dosage of pentobarbital (sleep onset 100%) and 25 mg/kg as sub-hypnotic dosage (sleep onset 0%). FA and DZP were administered (i.g.) 45 min prior to pentobarbital administration (i.p.). 5-HTP was injected (i.p.) 15 min prior to pentobarbital administration (i.p.). PCPA-pretreated mice received an injection of PCPA (300 mg/kg, s.c.) between 0800 and 0900, 24 h prior to the injection of pentobarbital. l-MA pretreated mice received an injection of l-MA (600 mg/kg) for 5 days between 0800 and 0900. On the fifth day, mice received l-MA 60 min prior to pentobarbital injection. Observers were blind to the drug treatment. Following pentobarbital administration, each mouse was observed for the sleep onset, a mouse losing righting reflex over 3 min was considered to be asleep. The time elapsed between pentobarbital injection and the loss of righting reflex was recorded as the sleep latency. The
Table 1 Effect of FA on locomotion activity in mice (n = 10). Groups
Locomotion activity (times/5 min)
Vehicle DZ 2.0 mg/kg FA 7.5 mg/kg FA 15.0 mg/kg FA 30.0 mg/kg
165.4 12.7 153.2 121.7 109.8
* **
± ± ± ± ±
12.7 6.8 16.3 17.6 15.5
Inhibitory (%) 0 92.3** 7.4 26.4* 33.6**
P < 0.05, compared with vehicle, ANOVA/SNK-test. P < 0.01, compared with vehicle, ANOVA/SNK-test.
time elapsed between the loss and recovery of the righting reflex was recorded as sleeping time. All data are presented as the mean ± S.E.M. For statistical comparisons, the results were analyzed by a one-way analysis of variance (ANOVA) followed by the Students–Newman–Keuls test (SNK) for post hoc comparisons. For the sub-hypnotic dosage of pentobarbital test, a chi-square test was used to compare the number of mice that fell asleep. P < 0.05 was considered statistically significant difference. Thirty minutes after FA administration, the locomotion activity of the mice was measured for 5 min. FA reduced the locomotion activity in a dose-dependent manner from 165.48 ± 12.7 (vehicle) to 153.2 ± 16.3 (7.5 mg/kg, P > 0.05), 121.7 ± 17.6 (15.0 mg/kg, P < 0.05), and 109.8 ± 15.5 (30.0 mg/kg, P < 0.01) (Table 1). The positive control DZP (2 mg/kg) also significantly decreased the locomotion activity (P < 0.01). FA showed synergistic effects with pentobarbital. In mice treated with the hypnotic dosage of pentobarbital (45 mg/kg), FA significantly shortened the sleep latency (Fig. 1A) and prolonged the sleeping time (Fig. 1B) in a dose-dependent manner at 15 mg/kg (P < 0.05) and 30 mg/kg (P < 0.01). In mice treated with the sub-hypnotic dosage of pentobarbital (25 mg/kg), FA significantly increased the rate of sleep onset in a dose-dependent manner
Fig. 1. Effect of FA on the hypnotic response to pentobarbital induced sleep in mice. The sleep latency (A) and the sleep time (B) were assessed. All data were presented as mean ± S.E.M. (n = 10 for vehicle; n = 15 for the other groups). *P < 0.05 and **P < 0.01 vs. vehicle (Student–Newman–Keuls test).
Y. Tu et al. / Neuroscience Letters 525 (2012) 95–99 Table 2 Effect of FA on sleep onset of mice induced by sub-hypnotic dosage of pentobarbital (25 mg/kg).
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Table 3 Synergic effects of FA with 5-HTP on sleep onset of mice treated with sub-hypnotic dosage of pentobarbital (25 mg/kg) (n = 15).
Groups
No. falling asleep/total no.
Sleep onset (%)
Groups
No. falling asleep/total no.
Sleep onset (%)
Vehicle DZ 2.0 mg/kg FA 7.5 mg/kg FA 15.0 mg/kg FA 30.0 mg/kg
0/15 10/10 2/15 5/15 10/15
0.0 100.0 13.3 33.3a 66.7b
Vehicle 2.5 mg/kg 5-HTP 7.5 mg/kg FA 2.5 mg/kg 5-HTP + 7.5 mg/kg FA
0/15 2/15 2/15 8/15
0.0 13.3a 13.3a 53.3b
a b
P < 0.05 vs. vehicle (chi-square test). P < 0.01 vs. vehicle (chi-square test).
at 15 mg/kg (P < 0.05) and 30 mg/kg (P < 0.01) (Table 2). As a positive control, DZP (2 mg/kg) also potentiated the hypnotic activity of pentobarbital in mice. FA administered alone did not induce sleep according to the present criterion. To investigate the relationship between the hypnotic activity of FA and the serotonergic system, the mice were treated with FA (7.5 mg/kg) for 45 min and with 5-HTP (2.5 mg/kg) for 15 min prior to the administration of pentobarbital (45 mg/kg, i.p.). Neither FA (7.5 mg/kg) nor 5-HTP (2.5 mg/kg) administered individually affected the sleep latency or sleeping time induced by the hypnotic dose of pentobarbital (Fig. 2A and B). The rate of sleep onset induced by the sub-hypnotic dosages of pentobarbital was also unaffected (Table 3). However, co-administration of FA (7.5 mg/kg) and 5-HTP (2.5 mg/kg) exhibited the synergistic effect of shortened sleep latency (P < 0.05, Fig. 2A) and prolonged sleeping time significantly (P < 0.05, Fig. 2B). The co-administration also significantly increased the rate of sleep onset (P < 0.05, Table 3) in mice treated with sub-hypnotic dosages of pentobarbital. Treatment with PCPA (300 mg/kg) has been shown to induce insomnia. In accordance with a previous report [3], the present
Fig. 2. Synergic effects of FA with 5-HTP on hypnotic response in pentobarbital treated mice (n = 15). Mice were administered FA (7.5 mg/kg) for 45 min prior to and 5-HTP (2.5 mg/kg) for 15 min prior to the injection of pentobarbital (45 mg/kg). The sleep latency (A) and the sleep time (B) were assessed. All data were presented as mean ± S.E.M. **P < 0.01 vs. vehicle. # P < 0.05 and ## P < 0.01 compared with 5-HTP (2.5 mg/kg) + FA (7.5 mg/kg) group (Student–Newman–Keuls test).
a b
P < 0.05 vs. 2.5 mg/kg 5-HTP + 2.5 mg/kg FA (chi-square test). P < 0.01 vs. vehicle (chi-square test).
study showed that mice pre-treated with PCPA (300 mg/kg, s.c.) for 24 h before pentobarbital injection significantly prolonged the sleep latency (P < 0.01, Fig. 3A) and shortened the sleeping time (P < 0.01, Fig. 3B) of pentobarbital induced sleep in mice. FA (30 mg/kg) significantly attenuated the insomnia effect of PCPA, which was reflected by decreased sleep latency (P < 0.01, Fig. 3A) and increased sleeping time (P < 0.01, Fig. 3B). A preliminary investigation showed that one-time treatment of l-MA did not affect pentobarbital-induced hypnosis (data not shown); consequently, we employed a continuous treatment model in which l-MA (600 mg/kg/day) was administered for 5 days. On the fifth day, the mice received l-MA (600 mg/kg) 60 min prior to the injection of pentobarbital (45 mg/kg). Pentobarbital-induced sleep was significantly abolished by l-MA, as reflected by increased sleep latency and decreased sleeping time. FA did not reverse the effect of l-MA on pentobarbital-induced sleep (Fig. 4). FA is a bioactive constituent of certain fruits, vegetables, and plant medicines. In the present study, the hypnotic-sedative activity of FA was evaluated. The results showed that FA exerted sedative effect by suppressing the locomotion activity in normal mice. FA also significantly potentiated the hypnotic activity of pentobarbital by shortening sleep latency (Fig. 1A), prolonging sleeping time (Fig. 1B), and by increasing the rate of sleep onset (Table 2).
Fig. 3. Effects of FA on PCPA-induced insomnia in pentobarbital treated mice (n = 15). Mice were pretreated with PCPA (300 mg/kg) for 24 h and FA for 45 min prior to the injection of pentobarbital (45 mg/kg). The sleep latency (A) and the sleep time (B) were assessed. All data were presented as mean ± S.E.M. **P < 0.01 vs. vehicle and ## P < 0.01 vs. group treated PCPA alone (Student–Newman–Keuls test).
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more than 95% of serotonin biosynthesis [13], resulting in complete insomnia or substantially reduced sleep [3,14,22]. These insomnia effects can be reversed by subsequent treatment with 5-HTP, which may restore serotonin biosynthesis, thereby restoring sleep [21,25]. This PCPA/5-HTP model suggests that 5-HT and 5-HTP are important for sleep regulation. The present study showed that acute treatment of PCPA 24 h prior to pentobarbital can induce sleep suppression and that this suppression can be inhibited by FA (Fig. 3). These results suggest that FA possesses hypnotic-sedative activity, which may be mediated by the serotonergic system. Previous studies of FA have focused on antioxidant-related bioactivities, such as inflammation, DNA damage [20], radiation protection [17], and anti-carcinogenic activities [1,11,16], among others. The present results suggest that FA may also be a candidate for treatment of insomnia and diseases that may induce sleep disorders. In summary, this work demonstrates that the serotonergic system may be involved in the hypnotic-sedative activity of FA. Further studies are needed to elucidate the hypnotic activity and mechanisms of FA and the relationship between FA and different 5-HT receptor subtypes. Acknowledgements This work was supported by the International Cooperation Program funded by Tianjin Municipal Science and Technology Commission (NO. 09ZCZDSF04600). Fig. 4. Effects of FA on the hypnotic reversing action of l-malic acid in pentobarbital treated mice (n = 15). Mice were pretreated with l-MA (600 mg/kg/day) for 5 days, on the fifth day, mice were administered l-MA for 60 min prior to and FA for 45 min prior to the injection of pentobarbital (45 mg/kg). The sleep latency (A) and the sleep time (B) were assessed. All data were presented as mean ± S.E.M. **P < 0.01 vs. vehicle and ## P < 0.01 vs. group treated l-MA alone (Student–Newman–Keuls test).
The sleep–wake cycle may be regulated by several different neural systems, including the GABAergic, serotonergic, histaminergic, and adrenergic systems [18]. The GABAergic system, which is the major sleep-promoting pathway, has been verified to play an important role in the action of many hypnotics. To investigate whether the hypnotic and sedative activities of FA were related to the GABAergic system, the effect of l-MA on the hypnotic activity of FA was evaluated. l-MA (a blocker of synthetic enzyme for GABA) alone did not potentiate pentobarbital-induced sleep; instead, it reversed the synergistic effect of other hypnotic agents on pentobarbital by inhibiting the synthesis of GABA and decreasing the concentration of GABA in the brain [23]. The present study showed that treatment with l-MA (600 mg/kg) alone did not affect the sleep of pentobarbital-treated mice; instead, it reversed the augmented effect of DZP (2.0 mg/kg) on pentobarbital-induced sleep (Fig. 4). FA (30 mg/kg) potentiated pentobarbital-induced sleep, and this effect was not decreased by l-MA (Fig. 4), suggesting that the potential effect of FA on pentobarbital-induced sleep may not be related to the GABAergic system. Serotonin is not only associated with the initiation and maintenance of sleep but also plays a role in inhibiting sleep and promoting wakefulness. The complex effects of serotonin on the sleep–wake cycle are attributed to serotonin acting at different brain sites associated with the control of sleep and wakefulness [8]. Previous studies have reported that different 5-HT receptors are selectively involved in the physiological functions [8,24]. 5-HTP, the precursor of 5-hydroxytryptamine (5-HT), prolongs pentobarbital-induced sleeping time in a dose-dependent manner [25]. The present study showed that FA produced synergistic effects with 5-HTP by potentiating pentobarbital-induced sleep in mice. Chronic administration of PCPA, a blocker of tryptophan hydroxylase, can inhibit
References [1] N. Baskaran, S. Manoharan, S. Balakrishnan, P. Pugalendhi, Chemopreventive potential of ferulic acid in 7,12-dimethylbenze[␣]anteracene-induced mammary carcinogenesis in Sprague-Dawley rats, Eur. J. Pharmacol. 637 (2010) 22–29. [2] M.L. Bocca, F. Le Doze, O. Etard, M. Pottier, J. L’Hoste, P. Denise, Residual effect of zolpidem 10 mg and zopiclone 7.5 mg versus flunitrazepam 1 mg and placebo on driving performance and ocular saccades, Psychopharmacology (Berl.) 143 (1999) 373–379. [3] A.A. Borbely, H.U. Neuhaus, I. Tobler, Effect of p-chlorophenylalanine and tryptophan on sleep, EEG and motor activity in the rat, Behav. Brain Res. 2 (1981) 1–22. [4] M.X. Chang, L.Y. Xu, J.S. Tao, Y. Feng, Study on the metabolism and pharmacokinetics of ferulic acid in rats, China Journal of Chinese Materia Medica 18 (1993) 300–302. [5] C.Y. Cheng, T.Y. Ho, E.J. Lee, S.Y. Su, N.Y. Tang, C.L. Hsieh, Ferulic acid reduces cerebral infarct through its antioxidative and anti-inflammatory effects following transient focal cerebral ischemia in rats, American Journal of Chinese Medicine 36 (2008) 1105–1119. [6] C.Y. Cheng, S.Y. Su, N.Y. Tang, T.Y. Ho, S.Y. Chiang, C.L. Hsieh, Ferulic acid provides neuroprotection against oxidative stress-related apoptosis after cerebral ischemia/reperfusion injury by inhibiting ICAM-1 mRNA expression in rats, Brain Research 1209 (2008) 136–150. [7] Q.P. Chu, L.E. Wang, X.Y. Cui, H.Z. Fu, Z.B. Lin, S.Q. Lin, Y.H. Zhang, Extract of Ganoderma lucidum potentiates pentobarbital-induced sleep via a GABAergic mechanism, Pharmacology Biochemistry and Behavior 86 (2007) 693–698. [8] C. Dugovic, Role of serotonin in sleep mechanisms, Revista de Neurologia 157 (2001) S16–S19. [9] A.N. Griffiths, D.M. Jones, A. Richens, Zopiclone produces effects on human performance similar to flurazepam, lormetazepam and triazolam, British Journal of Clinical Practice 21 (1986) 647–653. [10] F. Huang, Y. Xiong, L.H. Xu, S.P. Ma, C.G. Dou, Sedative and hypnotic activities of the ethanol fractions from Fructus Schisandrae in mice and rats, Journal of Ethnopharmacology 110 (2007) 471–475. [11] M.T. Huang, R.C. Smart, C.Q. Wong, A.H. Conney, Inhibitory effect of curcumin, chlorogenic acid, caffeic acid, and ferulic acid on promotion in mouse skin by 12-otetradecanoylphorbol-13-acetate, Cancer Res. 48 (1988) 5941–5946. [12] J. Kanski, M. Aksenova, A. Stoyanova, D.A. Butterfield, Ferulic acid antioxidant protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture systems in vitro: structure-activity studies, Journal of Nutritional Biochemistry 13 (2002) 273–281. [13] J.M. Khanna, H. Kalant, A.D. Le, J. Mayer, A.E. LeBlanc, Effects of pchlorophenylalanine on the acquisition of tolerance to the hypnotic effects of pentobarbital, barbital, and ethanol, Can. J. Physiol. Pharmacol. 58 (1980) 1031–1041. [14] W.P. Koella, A. Feldstein, J.S. Czicman, The effect of p-chlorophenylalanine on the sleep of cats, Electroencephalography and Clinical Neurophysiology 25 (1968) 481–490.
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Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Ferulic acid potentiates pentobarbital-induced sleep via the serotonergic system Yue Tu a,b , Shi-xiang Cheng a , Hong-tao Sun a , Tie-zhu Ma a , Sai Zhang a,∗ a b
Center for Neurology and Neurosurgery, Pingjin Hospital, Logistics College of Chinese People’s Armed Police Forces, Chenglin Road No. 220, Hedong District, Tianjin 300162, China Tianjin University of Traditional Chinese Medicine, Anshanxi Road No. 312, Nankai District, Tianjin 300193, China
h i g h l i g h t s
g r a p h i c a l
We investigated the sedative and hypnotic activities of ferulic acid. Ferulic acid inhibited the locomotion activity of mice. Ferulic acid potentiated the hypnotic effect of pentobarbital. Ferulic acid exerted sedative and hypnotic activity via the serotonergic system. Ferulic acid may be a pleasant candidate in therapy of insomnia.
Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a widespread natural phenolic compound which exert some bioactivity. The sedative and hypnotic effects of FA and possible mechanisms were investigated through behavioral pharmacology methods. The extract demonstrated that FA possessed sedative and hypnotic activities, which may mediated by serotonergic system.
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 20 March 2012 Received in revised form 23 July 2012 Accepted 29 July 2012 Keywords: Ferulic acid Sedative Hypnotic Pentobarbital Serotonergic system
a b s t r a c t
Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a widely distributed natural phenolic compound that is abundant in many plant tissues and foods. This study investigated possible mechanisms underlying the sedative–hypnotic effect of FA through behavioral pharmacology methods. FA showed dose-dependent sedative effects on locomotion activity in normal mice. FA also significantly potentiated pentobarbital-induced (45 mg/kg, i.p.) sleep by prolonging sleeping time and shortening sleep latency in a dose-dependent manner. These effects were augmented by the administration of 5hydroxytryptophan (5-HTP), a precursor of 5-hydroxytryptamine (5-HT). With a sub-hypnotic dose of pentobarbital (25 mg/kg, i.p.), FA significantly increased the rate of sleep onset and exhibited a synergistic effect with 5-HTP (2.5 mg/kg, i.p.). Pretreatment with p-chlorophenylalanine (PCPA, an inhibitor of tryptophan hydroxylase) significantly decreased the duration of pentobarbital-induced sleep, whereas FA significantly reversed this effect. These results suggest that FA has sedative–hypnotic activity, possibly mediated by the serotonergic system. © 2012 Elsevier Ireland Ltd. All rights reserved.
Insomnia, defined as persistent difficulty in initiating or maintaining sleep that affects daytime function, can induce significant psychological and physical disorders [10]. More than 27% of people worldwide experience insomnia, and approximately 3–10% of people are chronic and frequent users of hypnotics [7]. Clinically, benzodiazepines are the most widely used hypnotic agents
∗ Corresponding author. Tel.: +86 22 15216602576; fax: +86 22 60578627. E-mail address: [email protected] (S. Zhang). 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.07.068
prescribed for insomnia. However, benzodiazepines have many unpleasant side effects, including drug dependence, drug tolerance, rebound insomnia, and amnesia. New types of hypnotics, such as zolpidem and zolpiclone, also show some side effects [2,9]. Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a ubiquitous natural phenolic acid found in many plant tissues and foods, including Ferula assafoetida L., Ligusticum chuanxiong Hort, Cimicifuga foetida L., cabbages, wheat, rice bran, tomatoes, and onions. FA exhibits many bioactivities including antioxidant, antimicrobial, radio-protective, hypertensive, and anti-carcinogenic properties
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[1,11,16]. Currently, FA is widely used in the medical, food, and cosmetic industries. Because FA has low toxicity and is metabolized quickly, it has been approved as a food additive in some countries. FA can pass through the blood-brain barrier and reach the brain; its pharmacokinetics complies with the open one-compartment model in vivo. It can be absorbed completely and rapidly, metabolized quickly, and excreted by the kidneys without apparent accumulation in vivo [4]. FA exerts neuro-protective effects against oxidative stress-related apoptosis after cerebral ischemia and significantly reduces cerebral infarct in a transient middle cerebral artery occlusion (MCAO) model [5,6,15]. Additional studies have reported that FA can attenuate neuronal cell death caused by the uptake of oxidized low-density lipoprotein, hydroxyl and peroxyl radicals in vitro [12,19]. FA may also attenuate neuronal cell death during MCAO involved in inhibition of Akt signaling pathway inactivation and maintenance of the interaction between phosphor-Bad and 14-3-3 [15]. FA may also have sedative activity. However, to the best of our knowledge, no previous studies have reported any hypnotic-sedative activity of FA. The present study investigated the hypnotic effects and possible mechanisms of FA on pentobarbitalinduced sleep in rodents. A total of 390 male ICR mice (weight 18–22 g, Grade I; purchased from Tianjin Medical University) were used in this study. The mice were housed under controlled environmental conditions (temperature 22 ± 2 ◦ C, humidity 50 ± 10%, 12 h light/dark cycle and light on at 0600) and given ad libitum access to food and water. The mice were acclimated 1 week before testing. The mice were fasted for 12 h prior to the onset of the experiments. The experiments were carried out between 0800 and 1300 in a quiet room with temperatures ranging from 22 to 24 ◦ C. All procedures were conducted in accordance with the European Community guidelines for the use of experimental animals and approved by the Tianjin Medical University Committee on Animal Care and Use. For intragastric (i.g.) administration (0.2 ml/10 g, volume/body weight), FA (Sigma–Aldrich, St. Louis) and l-malic acid (l-MA) were dissolved and diazepam injection (DZP, 10 mg/2 ml, manufactured by People’s Pharmaceutical Manufacturer, Tianjin, China) was diluted with 0.5% dimethyl sulfoxide (DMSO). For intraperitoneal (i.p.) injection (0.1 ml/10 g, volume/body weight), 5-HTP (Alfa Aesar China Ltd., Beijing, China) and pentobarbital (Serve, Shanghai Chemical Reagent Corporation, China) were dissolved with physiological saline. For subcutaneous (s.c.) injection (0.1 ml/10 g, volume/body weight), PCPA (Sigma–Aldrich, St. Louis) was suspended in 0.5% gum acacia/physiological saline. The locomotion activity of mice was measured using an YLS1A Multi-autonomous Activity Instrument with five activity cages (Shandong Academy of Medical Sciences, Jinan, China) [23]. FA and DZP were administered orally (0.2 ml/10 g body weight). Twentyfive minutes after injection, mice were acclimated to the activity cages individually for 5 min; then, the locomotion activity of each mouse was measured for 5 min. The present study used 45 mg/kg as the hypnotic dosage of pentobarbital (sleep onset 100%) and 25 mg/kg as sub-hypnotic dosage (sleep onset 0%). FA and DZP were administered (i.g.) 45 min prior to pentobarbital administration (i.p.). 5-HTP was injected (i.p.) 15 min prior to pentobarbital administration (i.p.). PCPA-pretreated mice received an injection of PCPA (300 mg/kg, s.c.) between 0800 and 0900, 24 h prior to the injection of pentobarbital. l-MA pretreated mice received an injection of l-MA (600 mg/kg) for 5 days between 0800 and 0900. On the fifth day, mice received l-MA 60 min prior to pentobarbital injection. Observers were blind to the drug treatment. Following pentobarbital administration, each mouse was observed for the sleep onset, a mouse losing righting reflex over 3 min was considered to be asleep. The time elapsed between pentobarbital injection and the loss of righting reflex was recorded as the sleep latency. The
Table 1 Effect of FA on locomotion activity in mice (n = 10). Groups
Locomotion activity (times/5 min)
Vehicle DZ 2.0 mg/kg FA 7.5 mg/kg FA 15.0 mg/kg FA 30.0 mg/kg
165.4 12.7 153.2 121.7 109.8
* **
± ± ± ± ±
12.7 6.8 16.3 17.6 15.5
Inhibitory (%) 0 92.3** 7.4 26.4* 33.6**
P < 0.05, compared with vehicle, ANOVA/SNK-test. P < 0.01, compared with vehicle, ANOVA/SNK-test.
time elapsed between the loss and recovery of the righting reflex was recorded as sleeping time. All data are presented as the mean ± S.E.M. For statistical comparisons, the results were analyzed by a one-way analysis of variance (ANOVA) followed by the Students–Newman–Keuls test (SNK) for post hoc comparisons. For the sub-hypnotic dosage of pentobarbital test, a chi-square test was used to compare the number of mice that fell asleep. P < 0.05 was considered statistically significant difference. Thirty minutes after FA administration, the locomotion activity of the mice was measured for 5 min. FA reduced the locomotion activity in a dose-dependent manner from 165.48 ± 12.7 (vehicle) to 153.2 ± 16.3 (7.5 mg/kg, P > 0.05), 121.7 ± 17.6 (15.0 mg/kg, P < 0.05), and 109.8 ± 15.5 (30.0 mg/kg, P < 0.01) (Table 1). The positive control DZP (2 mg/kg) also significantly decreased the locomotion activity (P < 0.01). FA showed synergistic effects with pentobarbital. In mice treated with the hypnotic dosage of pentobarbital (45 mg/kg), FA significantly shortened the sleep latency (Fig. 1A) and prolonged the sleeping time (Fig. 1B) in a dose-dependent manner at 15 mg/kg (P < 0.05) and 30 mg/kg (P < 0.01). In mice treated with the sub-hypnotic dosage of pentobarbital (25 mg/kg), FA significantly increased the rate of sleep onset in a dose-dependent manner
Fig. 1. Effect of FA on the hypnotic response to pentobarbital induced sleep in mice. The sleep latency (A) and the sleep time (B) were assessed. All data were presented as mean ± S.E.M. (n = 10 for vehicle; n = 15 for the other groups). *P < 0.05 and **P < 0.01 vs. vehicle (Student–Newman–Keuls test).
Y. Tu et al. / Neuroscience Letters 525 (2012) 95–99 Table 2 Effect of FA on sleep onset of mice induced by sub-hypnotic dosage of pentobarbital (25 mg/kg).
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Table 3 Synergic effects of FA with 5-HTP on sleep onset of mice treated with sub-hypnotic dosage of pentobarbital (25 mg/kg) (n = 15).
Groups
No. falling asleep/total no.
Sleep onset (%)
Groups
No. falling asleep/total no.
Sleep onset (%)
Vehicle DZ 2.0 mg/kg FA 7.5 mg/kg FA 15.0 mg/kg FA 30.0 mg/kg
0/15 10/10 2/15 5/15 10/15
0.0 100.0 13.3 33.3a 66.7b
Vehicle 2.5 mg/kg 5-HTP 7.5 mg/kg FA 2.5 mg/kg 5-HTP + 7.5 mg/kg FA
0/15 2/15 2/15 8/15
0.0 13.3a 13.3a 53.3b
a b
P < 0.05 vs. vehicle (chi-square test). P < 0.01 vs. vehicle (chi-square test).
at 15 mg/kg (P < 0.05) and 30 mg/kg (P < 0.01) (Table 2). As a positive control, DZP (2 mg/kg) also potentiated the hypnotic activity of pentobarbital in mice. FA administered alone did not induce sleep according to the present criterion. To investigate the relationship between the hypnotic activity of FA and the serotonergic system, the mice were treated with FA (7.5 mg/kg) for 45 min and with 5-HTP (2.5 mg/kg) for 15 min prior to the administration of pentobarbital (45 mg/kg, i.p.). Neither FA (7.5 mg/kg) nor 5-HTP (2.5 mg/kg) administered individually affected the sleep latency or sleeping time induced by the hypnotic dose of pentobarbital (Fig. 2A and B). The rate of sleep onset induced by the sub-hypnotic dosages of pentobarbital was also unaffected (Table 3). However, co-administration of FA (7.5 mg/kg) and 5-HTP (2.5 mg/kg) exhibited the synergistic effect of shortened sleep latency (P < 0.05, Fig. 2A) and prolonged sleeping time significantly (P < 0.05, Fig. 2B). The co-administration also significantly increased the rate of sleep onset (P < 0.05, Table 3) in mice treated with sub-hypnotic dosages of pentobarbital. Treatment with PCPA (300 mg/kg) has been shown to induce insomnia. In accordance with a previous report [3], the present
Fig. 2. Synergic effects of FA with 5-HTP on hypnotic response in pentobarbital treated mice (n = 15). Mice were administered FA (7.5 mg/kg) for 45 min prior to and 5-HTP (2.5 mg/kg) for 15 min prior to the injection of pentobarbital (45 mg/kg). The sleep latency (A) and the sleep time (B) were assessed. All data were presented as mean ± S.E.M. **P < 0.01 vs. vehicle. # P < 0.05 and ## P < 0.01 compared with 5-HTP (2.5 mg/kg) + FA (7.5 mg/kg) group (Student–Newman–Keuls test).
a b
P < 0.05 vs. 2.5 mg/kg 5-HTP + 2.5 mg/kg FA (chi-square test). P < 0.01 vs. vehicle (chi-square test).
study showed that mice pre-treated with PCPA (300 mg/kg, s.c.) for 24 h before pentobarbital injection significantly prolonged the sleep latency (P < 0.01, Fig. 3A) and shortened the sleeping time (P < 0.01, Fig. 3B) of pentobarbital induced sleep in mice. FA (30 mg/kg) significantly attenuated the insomnia effect of PCPA, which was reflected by decreased sleep latency (P < 0.01, Fig. 3A) and increased sleeping time (P < 0.01, Fig. 3B). A preliminary investigation showed that one-time treatment of l-MA did not affect pentobarbital-induced hypnosis (data not shown); consequently, we employed a continuous treatment model in which l-MA (600 mg/kg/day) was administered for 5 days. On the fifth day, the mice received l-MA (600 mg/kg) 60 min prior to the injection of pentobarbital (45 mg/kg). Pentobarbital-induced sleep was significantly abolished by l-MA, as reflected by increased sleep latency and decreased sleeping time. FA did not reverse the effect of l-MA on pentobarbital-induced sleep (Fig. 4). FA is a bioactive constituent of certain fruits, vegetables, and plant medicines. In the present study, the hypnotic-sedative activity of FA was evaluated. The results showed that FA exerted sedative effect by suppressing the locomotion activity in normal mice. FA also significantly potentiated the hypnotic activity of pentobarbital by shortening sleep latency (Fig. 1A), prolonging sleeping time (Fig. 1B), and by increasing the rate of sleep onset (Table 2).
Fig. 3. Effects of FA on PCPA-induced insomnia in pentobarbital treated mice (n = 15). Mice were pretreated with PCPA (300 mg/kg) for 24 h and FA for 45 min prior to the injection of pentobarbital (45 mg/kg). The sleep latency (A) and the sleep time (B) were assessed. All data were presented as mean ± S.E.M. **P < 0.01 vs. vehicle and ## P < 0.01 vs. group treated PCPA alone (Student–Newman–Keuls test).
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more than 95% of serotonin biosynthesis [13], resulting in complete insomnia or substantially reduced sleep [3,14,22]. These insomnia effects can be reversed by subsequent treatment with 5-HTP, which may restore serotonin biosynthesis, thereby restoring sleep [21,25]. This PCPA/5-HTP model suggests that 5-HT and 5-HTP are important for sleep regulation. The present study showed that acute treatment of PCPA 24 h prior to pentobarbital can induce sleep suppression and that this suppression can be inhibited by FA (Fig. 3). These results suggest that FA possesses hypnotic-sedative activity, which may be mediated by the serotonergic system. Previous studies of FA have focused on antioxidant-related bioactivities, such as inflammation, DNA damage [20], radiation protection [17], and anti-carcinogenic activities [1,11,16], among others. The present results suggest that FA may also be a candidate for treatment of insomnia and diseases that may induce sleep disorders. In summary, this work demonstrates that the serotonergic system may be involved in the hypnotic-sedative activity of FA. Further studies are needed to elucidate the hypnotic activity and mechanisms of FA and the relationship between FA and different 5-HT receptor subtypes. Acknowledgements This work was supported by the International Cooperation Program funded by Tianjin Municipal Science and Technology Commission (NO. 09ZCZDSF04600). Fig. 4. Effects of FA on the hypnotic reversing action of l-malic acid in pentobarbital treated mice (n = 15). Mice were pretreated with l-MA (600 mg/kg/day) for 5 days, on the fifth day, mice were administered l-MA for 60 min prior to and FA for 45 min prior to the injection of pentobarbital (45 mg/kg). The sleep latency (A) and the sleep time (B) were assessed. All data were presented as mean ± S.E.M. **P < 0.01 vs. vehicle and ## P < 0.01 vs. group treated l-MA alone (Student–Newman–Keuls test).
The sleep–wake cycle may be regulated by several different neural systems, including the GABAergic, serotonergic, histaminergic, and adrenergic systems [18]. The GABAergic system, which is the major sleep-promoting pathway, has been verified to play an important role in the action of many hypnotics. To investigate whether the hypnotic and sedative activities of FA were related to the GABAergic system, the effect of l-MA on the hypnotic activity of FA was evaluated. l-MA (a blocker of synthetic enzyme for GABA) alone did not potentiate pentobarbital-induced sleep; instead, it reversed the synergistic effect of other hypnotic agents on pentobarbital by inhibiting the synthesis of GABA and decreasing the concentration of GABA in the brain [23]. The present study showed that treatment with l-MA (600 mg/kg) alone did not affect the sleep of pentobarbital-treated mice; instead, it reversed the augmented effect of DZP (2.0 mg/kg) on pentobarbital-induced sleep (Fig. 4). FA (30 mg/kg) potentiated pentobarbital-induced sleep, and this effect was not decreased by l-MA (Fig. 4), suggesting that the potential effect of FA on pentobarbital-induced sleep may not be related to the GABAergic system. Serotonin is not only associated with the initiation and maintenance of sleep but also plays a role in inhibiting sleep and promoting wakefulness. The complex effects of serotonin on the sleep–wake cycle are attributed to serotonin acting at different brain sites associated with the control of sleep and wakefulness [8]. Previous studies have reported that different 5-HT receptors are selectively involved in the physiological functions [8,24]. 5-HTP, the precursor of 5-hydroxytryptamine (5-HT), prolongs pentobarbital-induced sleeping time in a dose-dependent manner [25]. The present study showed that FA produced synergistic effects with 5-HTP by potentiating pentobarbital-induced sleep in mice. Chronic administration of PCPA, a blocker of tryptophan hydroxylase, can inhibit
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