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Antidepressant-like effect of genipin in mice
Neuroscience Letters 479 (2010) 236–239
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Antidepressant-like effect of genipin in mice Jun-Sheng Tian a,b , Yuan-Lu Cui a,b,∗ , Li-Min Hu a,b , Shan Gao a,b , Wei Chi a,b , Tian-Jiao Dong a,b , Lu-Ping Liu a,b a b
Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin, PR China Research Center of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, PR China
a r t i c l e
i n f o
Article history: Received 2 March 2010 Received in revised form 13 May 2010 Accepted 23 May 2010 Keywords: Genipin Antidepressant Reserpine Neurotransmitter Traditional Chinese Medicine
a b s t r a c t The present study aimed to investigate the antidepressant potential of genipin and its possible mechanisms. Mouse models of depression including the forced swimming test (FST) and the tail suspension test (TST) were used to evaluate the effects of genipin. A possible mechanism was explored in the test of antagonism of reserpine-induced ptosis and hypothermia in mice. The contents of monoamine neurotransmitters and their metabolites including epinephrine (NE), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in mice hippocampi were determined by HPLC–ECD. The results showed that intra-gastric administration of genipin at 50, 100, 200 mg/kg or fluoxetine at 7.5 mg/kg for 7 days significantly reduced the duration of immobility in FST and TST, while it did not affect the locomotor activity in the open field test (OFT). However, the effect was not dose-dependent. When the mice were treated with genipin or fluoxetine for 7 days, both of them could antagonize reserpine-induced ptosis and hypothermia. The 5-HT and NE contents in mice hippocampi were decreased after the peritoneal injection of reserpine at 2.0 mg/kg. The pre-treatment with genipin at 50, 100, 200 mg/kg or fluoxetine at 7.5 mg/kg for 7 days could elevate the contents of NE and 5-HT in mice hippocampi significantly. The results suggest that compared with fluoxetine, genipin exerts antidepressant-like effects significantly. A possible mechanism, at least in part, is the regulation of the 5-HT and NE levels in the hippocampus. © 2010 Elsevier Ireland Ltd. All rights reserved.
Herbal medicines have been the basis of health-care worldwide since the earliest days of human kind. Even in developed countries, patients rely on medicinal plants and herbal medicines for their primary care. In Germany, herbal medicines are fully recognized as medicines, whereas most herbal products in the USA are regulated as food or food supplements of dietary supplements, even though many are used by consumers as folk remedies [6]. In China and other oriental countries, there are lots of natural products such as peoniflorin, berberine, glycyrrhizin, rosmarinic acid, and hypericin that were involved with the treatment of depression disorders. In the present study, we put an emphasis on another natural compound, genipin, to investigate the potential antidepressant-like effects and its possible mechanisms on mice. Genipin is the aglycone of geniposide extracted from the fruit of Gardenia jasminoides Ellis. (Chinese herbal name is Zhi Zi), which has long been used for the treatment of diseases in China. The pharmacokinetics studies suggested that geniposide, when it is orally administered, hydrolyzed into genipin by enzymes produced by
∗ Corresponding author at: Research Center of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, No. 88, YuQuan Road, Nankai District, Tianjin, PR China. Tel.: +86 22 23051965. E-mail address: [email protected] (Y.-L. Cui). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.05.069
intestinal bacteria [1]. It is genipin, not geniposide that functions as the main bioactive compound to exhibit the pharmacological activities of gardenia [20]. Moreover, it has been proved that genipin not only reveals significant effects of anti-inflammatory and anti-angiogenesis, but also presents the abilities of inhibiting lipid peroxidation and production of nitrogen monoxide (NO), as well as protects hippocampal neurons from the toxicity of Alzhemer’ amyloid [7]. Yamazaki reported that genipin promoted neurite outgrowth in a dose-dependent manner [18] and Yamamoto et al. found that genipin could suppress in vitro fas-induced lethal liver apoptosis in mice [2]. Additionally, it was reported that genipin can increase the mitochondrial membrane potential, which then increase the ATP levels and close KATP channels, thereby stimulating insulin secretion [11]. According to the concepts of Traditional Chinese Medicine (TCM), the liver is one of the most vulnerable organs to anger, stress and depression resulting in stagnation of liver-qi (gan-qi-yujie), which were the etiological factors of depressive disorders [16]. In addition, many a Chinese Traditional Medicine such as Zhi-ZiChi-Tang, which was firstly described in “Treatise on Cold-induced Febride Diseases (Shang Han Lun)” written by Zhang Zhong Jing in 200–210 AD have been commonly used in folk for the therapeutic treatment of insomnia, deficient dysphoria. These major symptoms closely associated with the depressive disorders. However, direct
J.-S. Tian et al. / Neuroscience Letters 479 (2010) 236–239
evidence on the relationship between genipin and depression were not previously reported. Therefore, the present study was supposed to provide the details that genipin produced antidepressant-like effect and its possible mechanisms. The forced swimming test (FST) was a well-established animal model of depression widely used to screen new potential antidepressant drugs in rats and mice [12]. Another test was the tail suspension test (TST), which was conducted as previously described [15]. The forced swimming and tail suspension-induced state of immobility in animals claimed to represent a condition similar to human depression and amenable to reversal by antidepressant drugs [13]. These animal models were based on the despair or helplessness behavior to some inescapable and confined space in animals and are sensitive to various antidepressant drugs. These animal models play a role in the evaluation of antidepressant drugs. It is well known that monoamine neurotransmitters or its metabolite such as epinephrine (NE), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in central nervous system play a key role in the pathophysiology of depression and the alterations in the CNS are associated with the mechanisms of action underlying the therapeutic activity of antidepressant drugs [19]. Therefore, reversal of reserpine-induced ptosis and hypothermia in mice by sub-chronic treatment of genipin was performed in the present study to examine the monoamine mechanisms in mice hippocampus. Digibehave system (Jiliang Software Company, Shanghai, China) was used to record and analyze the animal behaviors. Agilent 1100 system with an electrochemical detector was used to determine the contents of neurotransmitters and its metabolite. Genipin (purity, 98%) was purchased from WAKO (Osaka, Japan) and NE, 5-HT, 5-HIAA, 1-octanesulfonic acid sodium salt (OSA) were all Sigmaaldrich (USA) products. Fluoxetine hydrochloride capsules were prescribed from the first affiliated hospital of Tianjin University of Traditional Chinese Medicine. All other chemical regents were of ACS or analytical grade. Male ICR strain of mice, weighing 18–20 g (Beijing Vital Laboratory Animal Technology Company, certificate number SCKX2009-0003) was used. All the animals were group-housed under a normal 12-h/12-h light/dark schedule with the lights on 08:00 a.m. and had free access to tap water and food pellets in a home cage of 30 cm × 15 cm × 10 cm. Ambient temperature and relative humidity were maintained at 24 ± 1 ◦ C and 55 ± 5%, respectively. The mice were acclimatized to the lab for 3–5 days before the experiment. All the experiments were carried out between 9:00 a.m. and 3:00 p.m. and the procedures performed in this study were in accordance with NIH Guide for the Care and Use of Laboratory Animals. In the FST, TST and OFT, single administration (p.o.) of genipin (50, 100 and 200 mg/kg), fluoxetine (7.5 mg/kg) or saline were performed 1 h prior to the test after a sustained treatment for 7 days. In the antagonism of reserpine-induced ptosis and hypothermia test, subchronic treatments of genipin (50, 100 and 200 mg/kg), fluoxetine (7.5 mg/kg) or saline were given to the mice for 7 days. Except the control group, mice were injected intraperitoneally with 2.0 mg/kg reserpine at 60 min later of the last treatment of drugs. The degree of ptosis and rectal temperature was determined at 60 and 120 min, respectively after the injection of reserpine. During the performance, food but not water, was withdrawn from the animals 2 h prior to the drug administration, and animals tested were blind to the observers. With the exception of the hippocampal monoamine analyses, different batches of mice were used in each test. The FST, established by Porsolt et al. [12], was one of the most widely used behavioral despair test. During the test, the mice were placed in a plexiglass cylinder (20 cm height ×10 cm diameter) containing 15 cm depth of water at 25 ± 1 ◦ C for 6 min. Duration of immobility was regarded as the time spent by the mouse floating in the water without struggling and making only those move-
237
ments necessary to keep its head above the water. The predominant behavior was assigned to be one of three categories: immobility, swimming and climbing. The whole process was recorded via double digital cameras and the lasted 4 min intervals of the 6 min test were analyzed by the Digibehave system. Decrease in the duration of immobility was considered to be a measurement of antidepressant activity [4]. The TST was firstly performed by Steru et al. [15]. Mice were individually hung 15 cm above the floor (1 cm from the tip of the end). The mice were thought to be immobile only when they hung passively and completely motionless. The behavior of the suspended mice was recorded and analyzed by Digibehave system, and the duration of immobility in the last 4 min was recorded and analyzed. In order to explore any association of immobility time in the FST and TST with the alterations in locomotor activity, mice treated with genipin or fluoxetine for 7 days were tested in the OFT [5]. The crossings and rearings were used to evaluate the activity. Briefly, each mice was placed individually into the center of an open field (50 cm × 50 cm × 40 cm) with blank surface covering the inside walls. The dark floor was divided into 25 equal squares marked by blank lines. The number of crossings and rearings were recorded by Digibehave system during a test period of last 3 min. The reserpine test was performed according to the method described by Bourin et al. [3]. After the last treatment of drugs, mice with a rectal temperature of 37–38 ◦ C were used. The degree of ptosis of each animal was recorded at 60 min after the injection of reserpine. Animals were placed on a shelf (20 cm above table top), and the degree of ptosis was rated according to the following rating scale: 0, eye open; 1, one-quarter closed; 2, half closed; 3, threequarters closed; 4, completely closed [14]. The rectal temperature of each animal was measured with a thermistor thermometer at 120 min after the injection of reserpine. Mice were sacrificed by decapitation after the final measurement of rectal temperatures. The bilateral hippocampi were removed from the brain and were put into the labeled tubes. A certain volume of ice-cold 0.1 M perchloric acid was added to each tube (1 mg: 10 L). The hippocampi were homogenized for 30 s and the homogenate was centrifuged at 14,000 × g for 20 min at 4 ◦ C. The resultant supernatant was injected to the HPLC instrument for the determination of monoamines. The HPLC–ECD conditions were as follow: Waters Symmetry C18 column (150 mm × 3.9 mm, 5 m) with a set of guard column; the mobile phase was 19% methanol, 3% acetonitrile and 78% phosphate buffer, where the buffer consisted of 0.1 M KH2 PO4 , 0.01 M OSA, 0.1 mM EDTA (pH: 3.6); the column temperature was at 35 ◦ C and the flow rate was 1.0 mL/min; the detector was set at a potential of +0.7 V relative to a Ag/AgCl reference electrode. Data were expressed as means ± SEM or means ± SD (n = 10). Single factor analysis of variance (ANOVA) technique was used to assess statistical significance of results when P < 0.01 or P < 0.05. Scheffe’s method was used for multiple comparison tests at level of 95%. The effects of oral administration of genipin on the duration of immobility in the mouse FST and TST were shown in Figs. 1 and 2, respectively. Treating animals with genipin for 7 days reduced the duration of immobility in FST significantly, especially at the doses of 50 and 100 mg/kg (P < 0.05). However, the decrease was not dose dependent in FST. The same treatment to the mice with genipin at doses of 50 and 100 mg/kg also decreased the immobility time significantly in TST. As a positive control, the classical antidepressant fluoxetine treated to the mice at daily dose of 7.5 mg/kg presented a marked reduction in immobility time both in FST and TST. In a result, treatment with genipin at 50 and 100 mg/kg or fluoxetine at 7.5 mg/kg which significantly reduced immobility time in FST and TST, did not increase the crossings and rearings in the OFT significantly(P > 0.05) (Table 1). It was an indication that the
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J.-S. Tian et al. / Neuroscience Letters 479 (2010) 236–239
Fig. 1. The effect of genipin (50, 100 and 200 mg/kg, p.o.) or fluoxetine (7.5 mg/kg, p.o.) on the duration of immobility in the forced swimming test. Values given are means ± SEM (n = 10). *P < 0.05, **P < 0.01 as compared with saline-treated control.
Fig. 2. The effect of genipin (50, 100 and 200 mg/kg, p.o.) or fluoxetine (7.5 mg/kg, p.o.) on the duration of immobility in the tail suspension test. Values given are means ± SEM (n = 10). *P < 0.05, **P < 0.01 as compared with saline-treated control.
locomotor activity in mice OFT was not affected by the treatment of genipin or fluoxetine. The effect of genipin or fluoxetine on reserpine-induced ptosis and hypothermia was shown in Table 2. The mice treated with only reserpine at the dose of 2.0 mg/kg presented significant alterations of ptosis and hypothermia compared with the control (# P < 0.05), and the pre-treatment of genipin at doses of 50 and 100 mg/kg or fluoxetine at dose of 7.5 mg/kg significantly antagonized reserpineinduced ptosis and hypothermia (**P < 0.01, *P < 0.05). However, genipin at the dose of 200 mg/kg only reversed the hypothermia but ptosis. Table 1 The effect of genipin (50, 100 and 200 mg/kg, p.o.) or fluoxetine (7.5 mg/kg, p.o.) on the crossings and rearings in the open-field test in mice. Groups
n
Dose (mg/kg)
Number of crossings
Control Genipin
10 10 10 10 10
– 50 100 200 7.5
145 125 123 131 134
Fluoxetine
± ± ± ± ±
29 32 32 37 33
Number of rearings 26 26 28 25 25
± ± ± ± ±
3 5 5 5 5
The crossings and rearings were calculated respectively and values given are means ± SD (n = 10), data were of no statistical significance.
Fig. 3. The effect of genipin (50, 100 and 200 mg/kg, p.o.) or fluoxetine (7.5 mg/kg, p.o.) on the concentration of monoamine neurotransmitters in the reserpineinduced mice hippocampi. Values given are means ± SEM (n = 10). # P < 0.05 as compared with the saline-treated control, and *P < 0.05, **P < 0.01 as compared with the reserpine group.
Results from the HPLC–ECD determination of monoamines in mice hippocampi were shown in Fig. 3. The mice treated with reserpine at dose of 2.0 mg/kg significantly reduce the contents of NE, 5-HT and 5-HIAA (# P < 0.05), while the pre-treatment of genipin or fluoxetine significantly elevated the contents of NE and 5-HT (**P < 0.01, *P < 0.05). The contents of 5-HIAA were increased but without statistical significance. The similar tendency of the results between the behavior test and reserpine test was presented that the effect was not dose-dependent. The classical antidepressants include the tricyclic antidepressant (TCA), monoamine oxidase inhibitor (MAOI), and selective serotonin reuptake inhibitor (SSRI). Fluoxetine, selected as the positive control in the present study, is one of the most commonly and widely used well-established clinical antidepressant. Although these drugs show excellent efficacy, most of them frequently produce adverse effects. What an indication is that the relative effective and safe antidepressant drugs were expected sincerely and the discovery of novel antidepressant drugs were gradually being paid more attentions to. For example, Hypericum perforatum L. (St John’s Wort), as a perennial plant, is the only herbal alternative to classic synthetic antidepressants in the therapy of mild to moderate depression [9]. As widely used animal model for assessing pharmacological antidepressant-like effects, FST and TST were introduced. In this test, mice were under stress from which they could not escape. After an initial period of struggling, they would become immobile, resembling a state of despair and mental depression. The main indication of the antidepressant-like action of any given compound is the marked reduction in the duration of immobility. In the present study, genipin or fluoxetine were treated to mice orally to investigate the antidepressant-like effects, and the duration of immobility reduced markedly. As a result, genipin produced antidepressant-like effect broadly similar to the fluoxetine. On the other hand, genipin did not affect the locomotor activity in the OFT, indicating that antidepressant actions of genipin in mouse FST and TST seemed unlikely to be due to an increase in locomotor activity. Besides FST and TST, other involved animal models of depression including learned helplessness, olfactory bulbectomy model, reserpin-induced model and chronic mild stress (CMS) model. In them, the CMS model plays a role in the evaluation of the depression, which is involved with repeated exposure to relatively moderate stress in a period of 21 or 28 days [10].
J.-S. Tian et al. / Neuroscience Letters 479 (2010) 236–239
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Table 2 The antagonism of genipin (50, 100 and 200 mg/kg, p.o.) or fluoxetine (7.5 mg/kg, p.o.) on the reserpine-induced ptosis and hypothermia in mice (n = 10). Groups
Dose (mg/kg)
Reserpine (2.0 mg/kg)
Mean score of ptosis
Rectal temperature (◦ C)
Control Reserpine Genipin
– – 50 100 200 7.5
– + + + + +
0 2.5 ± 0.5# 1.5 ± 1.0** 1.9 ± 0.9* 2.3 ± 0.7 1.3 ± 0.7**
37.1 35.7 36.7 36.4 36.1 36.7
Fluoxetine
± ± ± ± ± ±
0.6 0.7# 0.2** 0.4** 0.1** 0.3**
The mean score of ptosis and rectal temperature was calculated respectively and values were given as means ± SD with 10 mice each group. * P < 0.05 as compared with the group of reserpine. ** P < 0.01 as compared with the group of reserpine. # P < 0.05 as compared with the group of control.
Depressive disorder has been associated with disturbances of brain monoamine neurotransmitters. Therefore, we investigated the possible antidepressive mechanism of genipin on the reserpine-induced animal depression model which is based on the monoamine hypothesis of depression [8]. Reserpine can irreversibly inhibit the vesicular uptake of monoamines, including noradrenaline, dopamine and 5-hydroxytrytamine and its metabolites. Consequently, ptosis and hypothermia are observed as depletion of monoamines stores or stimulation of monoamines reuptake. Genipin significantly antagonized the reserpine-induced ptosis and hypothermia as well as the contents of NE and 5-HT in mice hippocampi. These results suggested that the antidepressant effect of genipin was likely to be mediated through central monoaminergic neurotransmitter system. However, as the treatment with genipin at the dose of 200 mg/kg did not reverse the ptosis, the degree of monoamines or its metabolite did not alter in a dose dependent fashion and the antidepressant-like effect was also non-dose dependent, which were an indication that other mechanisms at post-synaptic level such as NO-cGMP-PKG signaling pathway may be involved [17]. Treatment with genipin at 25 mg/kg had been done in the FST and TST, while the duration of immobility was of no statistical significance compared with the control. The antidepressant potential of genipin in the chrionic mild unpredicted stress (CMS) rat depression model is performing now, and additional research is under way to explore the other possible mechanisms of the antidepressant-like effect of genipin. In a conclusion, genipin exerts antidepressant-like effect in the FST and TST in mice. The results demonstrated that the antidepressant-like effect of genipin is mediated, at least in part, via the central monoaminergic neurotransmitter system. Acknowledgments This work was supported by National Natural Science Foundation of China (30973967) and Specialized Research Fund for the Doctoral Program of Higher Education (20091210110003). References [1] T. Akao, K. Kobayashi, M. Aburada, Enzymic studies on the animal and intestinal bacterial metabolism of geniposide, Biol. Phar. Bull. 17 (1994) 1573–1576.
[2] M. Yamamoto, N. Miura, N. Ohtake, S. Amagya, Genipin, a metabolite derived from the herbal inchin-ko-to, and suppression of fas-induced lethal liver apoptosis in mice, Gastroenterology 118 (2000) 380–389. [3] M Bourin, M. Poncelet, R. Chermat, P. Simon, The value of the reserpine test in psychopharmacology, Arzneimittel-Forschung 33 (1983) 1173–1176. [4] J.F. Cryan, A. Holmes, The ascent of mouse: advances in modeling human depression and anxiety, Nutr. Rev. 4 (2005) 775–790. [5] M.R. Herrera, S. Mora, G.S. Viana, Antidepressant and anxiolytic effects of hydroalcoholic extract from Salvia elegans, J. Ethnopharmacol. 107 (2006) 53–58. [6] E.T. Kleber, S. Obry, W. Hippeli, E.F. Elstner, Biochemical activities of extracts from Hypericum perforatum L., Arzneimittel-Forschung 49 (1999) 106– 109. [7] H.J. Koo, Y.S. Song, H.J. Kim, E.H. Park, Antiinflammatory effects of genipin, an active principle of gardenia, Eur. J. Pharmacol. 495 (2004) 201–208. [8] Q.Q. Mao, Z. Huang, Antidepressant drugs and animal models of depression, Foreign Medical Sciences, Sec. Psychiatry 32 (2005) 216–220. [9] W. Mario, S.Z. Manfred, Hypericum perforatum: a modern herbal antidepressant, Clin. Pharmacokinet. 45 (2006) 449–468. [10] M.J. Millan, The role of monamines in the actions of established and novel antidepressant agents: a critical review, Eur. J. Pharmacol. 500 (2004) 371– 384. [11] L.E. Parton, C.P. Ye, R. Coppari, Glucose sensing by POMC neurons regulates glucose homeostasis and is impaired in obesity, Nature 449 (2007) 228– 233. [12] R.D. Porsolt, M. Jalfre, A. Bertin, Behavioral despair in mice: a primary screening test for antidepressants, Arch. Int. Pharmacodyn. Ther. 229 (1977) 326– 327. [13] C.E. Renard, E. Dailly, D.J. David, Monoamine metabolism changes following the mouse forced swimming test but not the tail suspension test, Fundam. Clin. Pharmacol. 17 (2003) 449–455. [14] C.C. Sánchez-Mateo, C.X. Bonkanka, B. Prado, R.M. Rabanal, Antidepressant activity of some Hypericum reflexum L. Fil. extracts in the forced swimming test in mice, J. Ethnopharmacol. 112 (2007) 115–121. [15] L. Steru, R. Chermat, B. Thierry, The tail suspension test: a new method for screening antidepressants in mice, Psychopharmacology (Berl.) 85 (1985) 367–370. [16] Y.T. Wang, Q.R. Tan, L.L. Sun, J. Cao, K.F. Dou, B. Xia, W. Wang, Possible therapeutic effect of a Traditional Chinese Medicine, si-ni-san, on chronic restraint stress related disorders, Neurosci. Lett. 449 (2009) 215–219. [17] M Yamazaki, K. Chiba, Genipin exhibits neurotrophic effects through a common signaling pathway in nitric oxide synthase-expressing cell, Eur. J. Pharmacol. 581 (2008) 255–261. [18] M. Yamazaki, K. Chiba, T. Mohri, Neuritogenic effect of natural irioid compounds on PC12h cells and its possible relation to signaling protein kinases, Biol. Phar. Bull. 19 (1996) 791–795. [19] L.T. Yi, Y.C. Li, Y. Pan, J.M. Li, H.F. Kung, Antidepressant-like effects of psoralidin isolated from the seeds of Psoralea corylifolia in the forced swimming test in mice, Prog. Neuropsychopharmacol. Biol. Psychiatry 32 (2008) 510– 519. [20] H.Z. Zheng, Z.H. Dong, J. Yu, Modern Research and Application of Chinese Traditional Medicine, vol.4, Academy Press, Beijing, China, 2000, 3166 pp.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Antidepressant-like effect of genipin in mice Jun-Sheng Tian a,b , Yuan-Lu Cui a,b,∗ , Li-Min Hu a,b , Shan Gao a,b , Wei Chi a,b , Tian-Jiao Dong a,b , Lu-Ping Liu a,b a b
Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin, PR China Research Center of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, PR China
a r t i c l e
i n f o
Article history: Received 2 March 2010 Received in revised form 13 May 2010 Accepted 23 May 2010 Keywords: Genipin Antidepressant Reserpine Neurotransmitter Traditional Chinese Medicine
a b s t r a c t The present study aimed to investigate the antidepressant potential of genipin and its possible mechanisms. Mouse models of depression including the forced swimming test (FST) and the tail suspension test (TST) were used to evaluate the effects of genipin. A possible mechanism was explored in the test of antagonism of reserpine-induced ptosis and hypothermia in mice. The contents of monoamine neurotransmitters and their metabolites including epinephrine (NE), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in mice hippocampi were determined by HPLC–ECD. The results showed that intra-gastric administration of genipin at 50, 100, 200 mg/kg or fluoxetine at 7.5 mg/kg for 7 days significantly reduced the duration of immobility in FST and TST, while it did not affect the locomotor activity in the open field test (OFT). However, the effect was not dose-dependent. When the mice were treated with genipin or fluoxetine for 7 days, both of them could antagonize reserpine-induced ptosis and hypothermia. The 5-HT and NE contents in mice hippocampi were decreased after the peritoneal injection of reserpine at 2.0 mg/kg. The pre-treatment with genipin at 50, 100, 200 mg/kg or fluoxetine at 7.5 mg/kg for 7 days could elevate the contents of NE and 5-HT in mice hippocampi significantly. The results suggest that compared with fluoxetine, genipin exerts antidepressant-like effects significantly. A possible mechanism, at least in part, is the regulation of the 5-HT and NE levels in the hippocampus. © 2010 Elsevier Ireland Ltd. All rights reserved.
Herbal medicines have been the basis of health-care worldwide since the earliest days of human kind. Even in developed countries, patients rely on medicinal plants and herbal medicines for their primary care. In Germany, herbal medicines are fully recognized as medicines, whereas most herbal products in the USA are regulated as food or food supplements of dietary supplements, even though many are used by consumers as folk remedies [6]. In China and other oriental countries, there are lots of natural products such as peoniflorin, berberine, glycyrrhizin, rosmarinic acid, and hypericin that were involved with the treatment of depression disorders. In the present study, we put an emphasis on another natural compound, genipin, to investigate the potential antidepressant-like effects and its possible mechanisms on mice. Genipin is the aglycone of geniposide extracted from the fruit of Gardenia jasminoides Ellis. (Chinese herbal name is Zhi Zi), which has long been used for the treatment of diseases in China. The pharmacokinetics studies suggested that geniposide, when it is orally administered, hydrolyzed into genipin by enzymes produced by
∗ Corresponding author at: Research Center of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, No. 88, YuQuan Road, Nankai District, Tianjin, PR China. Tel.: +86 22 23051965. E-mail address: [email protected] (Y.-L. Cui). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.05.069
intestinal bacteria [1]. It is genipin, not geniposide that functions as the main bioactive compound to exhibit the pharmacological activities of gardenia [20]. Moreover, it has been proved that genipin not only reveals significant effects of anti-inflammatory and anti-angiogenesis, but also presents the abilities of inhibiting lipid peroxidation and production of nitrogen monoxide (NO), as well as protects hippocampal neurons from the toxicity of Alzhemer’ amyloid [7]. Yamazaki reported that genipin promoted neurite outgrowth in a dose-dependent manner [18] and Yamamoto et al. found that genipin could suppress in vitro fas-induced lethal liver apoptosis in mice [2]. Additionally, it was reported that genipin can increase the mitochondrial membrane potential, which then increase the ATP levels and close KATP channels, thereby stimulating insulin secretion [11]. According to the concepts of Traditional Chinese Medicine (TCM), the liver is one of the most vulnerable organs to anger, stress and depression resulting in stagnation of liver-qi (gan-qi-yujie), which were the etiological factors of depressive disorders [16]. In addition, many a Chinese Traditional Medicine such as Zhi-ZiChi-Tang, which was firstly described in “Treatise on Cold-induced Febride Diseases (Shang Han Lun)” written by Zhang Zhong Jing in 200–210 AD have been commonly used in folk for the therapeutic treatment of insomnia, deficient dysphoria. These major symptoms closely associated with the depressive disorders. However, direct
J.-S. Tian et al. / Neuroscience Letters 479 (2010) 236–239
evidence on the relationship between genipin and depression were not previously reported. Therefore, the present study was supposed to provide the details that genipin produced antidepressant-like effect and its possible mechanisms. The forced swimming test (FST) was a well-established animal model of depression widely used to screen new potential antidepressant drugs in rats and mice [12]. Another test was the tail suspension test (TST), which was conducted as previously described [15]. The forced swimming and tail suspension-induced state of immobility in animals claimed to represent a condition similar to human depression and amenable to reversal by antidepressant drugs [13]. These animal models were based on the despair or helplessness behavior to some inescapable and confined space in animals and are sensitive to various antidepressant drugs. These animal models play a role in the evaluation of antidepressant drugs. It is well known that monoamine neurotransmitters or its metabolite such as epinephrine (NE), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in central nervous system play a key role in the pathophysiology of depression and the alterations in the CNS are associated with the mechanisms of action underlying the therapeutic activity of antidepressant drugs [19]. Therefore, reversal of reserpine-induced ptosis and hypothermia in mice by sub-chronic treatment of genipin was performed in the present study to examine the monoamine mechanisms in mice hippocampus. Digibehave system (Jiliang Software Company, Shanghai, China) was used to record and analyze the animal behaviors. Agilent 1100 system with an electrochemical detector was used to determine the contents of neurotransmitters and its metabolite. Genipin (purity, 98%) was purchased from WAKO (Osaka, Japan) and NE, 5-HT, 5-HIAA, 1-octanesulfonic acid sodium salt (OSA) were all Sigmaaldrich (USA) products. Fluoxetine hydrochloride capsules were prescribed from the first affiliated hospital of Tianjin University of Traditional Chinese Medicine. All other chemical regents were of ACS or analytical grade. Male ICR strain of mice, weighing 18–20 g (Beijing Vital Laboratory Animal Technology Company, certificate number SCKX2009-0003) was used. All the animals were group-housed under a normal 12-h/12-h light/dark schedule with the lights on 08:00 a.m. and had free access to tap water and food pellets in a home cage of 30 cm × 15 cm × 10 cm. Ambient temperature and relative humidity were maintained at 24 ± 1 ◦ C and 55 ± 5%, respectively. The mice were acclimatized to the lab for 3–5 days before the experiment. All the experiments were carried out between 9:00 a.m. and 3:00 p.m. and the procedures performed in this study were in accordance with NIH Guide for the Care and Use of Laboratory Animals. In the FST, TST and OFT, single administration (p.o.) of genipin (50, 100 and 200 mg/kg), fluoxetine (7.5 mg/kg) or saline were performed 1 h prior to the test after a sustained treatment for 7 days. In the antagonism of reserpine-induced ptosis and hypothermia test, subchronic treatments of genipin (50, 100 and 200 mg/kg), fluoxetine (7.5 mg/kg) or saline were given to the mice for 7 days. Except the control group, mice were injected intraperitoneally with 2.0 mg/kg reserpine at 60 min later of the last treatment of drugs. The degree of ptosis and rectal temperature was determined at 60 and 120 min, respectively after the injection of reserpine. During the performance, food but not water, was withdrawn from the animals 2 h prior to the drug administration, and animals tested were blind to the observers. With the exception of the hippocampal monoamine analyses, different batches of mice were used in each test. The FST, established by Porsolt et al. [12], was one of the most widely used behavioral despair test. During the test, the mice were placed in a plexiglass cylinder (20 cm height ×10 cm diameter) containing 15 cm depth of water at 25 ± 1 ◦ C for 6 min. Duration of immobility was regarded as the time spent by the mouse floating in the water without struggling and making only those move-
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ments necessary to keep its head above the water. The predominant behavior was assigned to be one of three categories: immobility, swimming and climbing. The whole process was recorded via double digital cameras and the lasted 4 min intervals of the 6 min test were analyzed by the Digibehave system. Decrease in the duration of immobility was considered to be a measurement of antidepressant activity [4]. The TST was firstly performed by Steru et al. [15]. Mice were individually hung 15 cm above the floor (1 cm from the tip of the end). The mice were thought to be immobile only when they hung passively and completely motionless. The behavior of the suspended mice was recorded and analyzed by Digibehave system, and the duration of immobility in the last 4 min was recorded and analyzed. In order to explore any association of immobility time in the FST and TST with the alterations in locomotor activity, mice treated with genipin or fluoxetine for 7 days were tested in the OFT [5]. The crossings and rearings were used to evaluate the activity. Briefly, each mice was placed individually into the center of an open field (50 cm × 50 cm × 40 cm) with blank surface covering the inside walls. The dark floor was divided into 25 equal squares marked by blank lines. The number of crossings and rearings were recorded by Digibehave system during a test period of last 3 min. The reserpine test was performed according to the method described by Bourin et al. [3]. After the last treatment of drugs, mice with a rectal temperature of 37–38 ◦ C were used. The degree of ptosis of each animal was recorded at 60 min after the injection of reserpine. Animals were placed on a shelf (20 cm above table top), and the degree of ptosis was rated according to the following rating scale: 0, eye open; 1, one-quarter closed; 2, half closed; 3, threequarters closed; 4, completely closed [14]. The rectal temperature of each animal was measured with a thermistor thermometer at 120 min after the injection of reserpine. Mice were sacrificed by decapitation after the final measurement of rectal temperatures. The bilateral hippocampi were removed from the brain and were put into the labeled tubes. A certain volume of ice-cold 0.1 M perchloric acid was added to each tube (1 mg: 10 L). The hippocampi were homogenized for 30 s and the homogenate was centrifuged at 14,000 × g for 20 min at 4 ◦ C. The resultant supernatant was injected to the HPLC instrument for the determination of monoamines. The HPLC–ECD conditions were as follow: Waters Symmetry C18 column (150 mm × 3.9 mm, 5 m) with a set of guard column; the mobile phase was 19% methanol, 3% acetonitrile and 78% phosphate buffer, where the buffer consisted of 0.1 M KH2 PO4 , 0.01 M OSA, 0.1 mM EDTA (pH: 3.6); the column temperature was at 35 ◦ C and the flow rate was 1.0 mL/min; the detector was set at a potential of +0.7 V relative to a Ag/AgCl reference electrode. Data were expressed as means ± SEM or means ± SD (n = 10). Single factor analysis of variance (ANOVA) technique was used to assess statistical significance of results when P < 0.01 or P < 0.05. Scheffe’s method was used for multiple comparison tests at level of 95%. The effects of oral administration of genipin on the duration of immobility in the mouse FST and TST were shown in Figs. 1 and 2, respectively. Treating animals with genipin for 7 days reduced the duration of immobility in FST significantly, especially at the doses of 50 and 100 mg/kg (P < 0.05). However, the decrease was not dose dependent in FST. The same treatment to the mice with genipin at doses of 50 and 100 mg/kg also decreased the immobility time significantly in TST. As a positive control, the classical antidepressant fluoxetine treated to the mice at daily dose of 7.5 mg/kg presented a marked reduction in immobility time both in FST and TST. In a result, treatment with genipin at 50 and 100 mg/kg or fluoxetine at 7.5 mg/kg which significantly reduced immobility time in FST and TST, did not increase the crossings and rearings in the OFT significantly(P > 0.05) (Table 1). It was an indication that the
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Fig. 1. The effect of genipin (50, 100 and 200 mg/kg, p.o.) or fluoxetine (7.5 mg/kg, p.o.) on the duration of immobility in the forced swimming test. Values given are means ± SEM (n = 10). *P < 0.05, **P < 0.01 as compared with saline-treated control.
Fig. 2. The effect of genipin (50, 100 and 200 mg/kg, p.o.) or fluoxetine (7.5 mg/kg, p.o.) on the duration of immobility in the tail suspension test. Values given are means ± SEM (n = 10). *P < 0.05, **P < 0.01 as compared with saline-treated control.
locomotor activity in mice OFT was not affected by the treatment of genipin or fluoxetine. The effect of genipin or fluoxetine on reserpine-induced ptosis and hypothermia was shown in Table 2. The mice treated with only reserpine at the dose of 2.0 mg/kg presented significant alterations of ptosis and hypothermia compared with the control (# P < 0.05), and the pre-treatment of genipin at doses of 50 and 100 mg/kg or fluoxetine at dose of 7.5 mg/kg significantly antagonized reserpineinduced ptosis and hypothermia (**P < 0.01, *P < 0.05). However, genipin at the dose of 200 mg/kg only reversed the hypothermia but ptosis. Table 1 The effect of genipin (50, 100 and 200 mg/kg, p.o.) or fluoxetine (7.5 mg/kg, p.o.) on the crossings and rearings in the open-field test in mice. Groups
n
Dose (mg/kg)
Number of crossings
Control Genipin
10 10 10 10 10
– 50 100 200 7.5
145 125 123 131 134
Fluoxetine
± ± ± ± ±
29 32 32 37 33
Number of rearings 26 26 28 25 25
± ± ± ± ±
3 5 5 5 5
The crossings and rearings were calculated respectively and values given are means ± SD (n = 10), data were of no statistical significance.
Fig. 3. The effect of genipin (50, 100 and 200 mg/kg, p.o.) or fluoxetine (7.5 mg/kg, p.o.) on the concentration of monoamine neurotransmitters in the reserpineinduced mice hippocampi. Values given are means ± SEM (n = 10). # P < 0.05 as compared with the saline-treated control, and *P < 0.05, **P < 0.01 as compared with the reserpine group.
Results from the HPLC–ECD determination of monoamines in mice hippocampi were shown in Fig. 3. The mice treated with reserpine at dose of 2.0 mg/kg significantly reduce the contents of NE, 5-HT and 5-HIAA (# P < 0.05), while the pre-treatment of genipin or fluoxetine significantly elevated the contents of NE and 5-HT (**P < 0.01, *P < 0.05). The contents of 5-HIAA were increased but without statistical significance. The similar tendency of the results between the behavior test and reserpine test was presented that the effect was not dose-dependent. The classical antidepressants include the tricyclic antidepressant (TCA), monoamine oxidase inhibitor (MAOI), and selective serotonin reuptake inhibitor (SSRI). Fluoxetine, selected as the positive control in the present study, is one of the most commonly and widely used well-established clinical antidepressant. Although these drugs show excellent efficacy, most of them frequently produce adverse effects. What an indication is that the relative effective and safe antidepressant drugs were expected sincerely and the discovery of novel antidepressant drugs were gradually being paid more attentions to. For example, Hypericum perforatum L. (St John’s Wort), as a perennial plant, is the only herbal alternative to classic synthetic antidepressants in the therapy of mild to moderate depression [9]. As widely used animal model for assessing pharmacological antidepressant-like effects, FST and TST were introduced. In this test, mice were under stress from which they could not escape. After an initial period of struggling, they would become immobile, resembling a state of despair and mental depression. The main indication of the antidepressant-like action of any given compound is the marked reduction in the duration of immobility. In the present study, genipin or fluoxetine were treated to mice orally to investigate the antidepressant-like effects, and the duration of immobility reduced markedly. As a result, genipin produced antidepressant-like effect broadly similar to the fluoxetine. On the other hand, genipin did not affect the locomotor activity in the OFT, indicating that antidepressant actions of genipin in mouse FST and TST seemed unlikely to be due to an increase in locomotor activity. Besides FST and TST, other involved animal models of depression including learned helplessness, olfactory bulbectomy model, reserpin-induced model and chronic mild stress (CMS) model. In them, the CMS model plays a role in the evaluation of the depression, which is involved with repeated exposure to relatively moderate stress in a period of 21 or 28 days [10].
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Table 2 The antagonism of genipin (50, 100 and 200 mg/kg, p.o.) or fluoxetine (7.5 mg/kg, p.o.) on the reserpine-induced ptosis and hypothermia in mice (n = 10). Groups
Dose (mg/kg)
Reserpine (2.0 mg/kg)
Mean score of ptosis
Rectal temperature (◦ C)
Control Reserpine Genipin
– – 50 100 200 7.5
– + + + + +
0 2.5 ± 0.5# 1.5 ± 1.0** 1.9 ± 0.9* 2.3 ± 0.7 1.3 ± 0.7**
37.1 35.7 36.7 36.4 36.1 36.7
Fluoxetine
± ± ± ± ± ±
0.6 0.7# 0.2** 0.4** 0.1** 0.3**
The mean score of ptosis and rectal temperature was calculated respectively and values were given as means ± SD with 10 mice each group. * P < 0.05 as compared with the group of reserpine. ** P < 0.01 as compared with the group of reserpine. # P < 0.05 as compared with the group of control.
Depressive disorder has been associated with disturbances of brain monoamine neurotransmitters. Therefore, we investigated the possible antidepressive mechanism of genipin on the reserpine-induced animal depression model which is based on the monoamine hypothesis of depression [8]. Reserpine can irreversibly inhibit the vesicular uptake of monoamines, including noradrenaline, dopamine and 5-hydroxytrytamine and its metabolites. Consequently, ptosis and hypothermia are observed as depletion of monoamines stores or stimulation of monoamines reuptake. Genipin significantly antagonized the reserpine-induced ptosis and hypothermia as well as the contents of NE and 5-HT in mice hippocampi. These results suggested that the antidepressant effect of genipin was likely to be mediated through central monoaminergic neurotransmitter system. However, as the treatment with genipin at the dose of 200 mg/kg did not reverse the ptosis, the degree of monoamines or its metabolite did not alter in a dose dependent fashion and the antidepressant-like effect was also non-dose dependent, which were an indication that other mechanisms at post-synaptic level such as NO-cGMP-PKG signaling pathway may be involved [17]. Treatment with genipin at 25 mg/kg had been done in the FST and TST, while the duration of immobility was of no statistical significance compared with the control. The antidepressant potential of genipin in the chrionic mild unpredicted stress (CMS) rat depression model is performing now, and additional research is under way to explore the other possible mechanisms of the antidepressant-like effect of genipin. In a conclusion, genipin exerts antidepressant-like effect in the FST and TST in mice. The results demonstrated that the antidepressant-like effect of genipin is mediated, at least in part, via the central monoaminergic neurotransmitter system. Acknowledgments This work was supported by National Natural Science Foundation of China (30973967) and Specialized Research Fund for the Doctoral Program of Higher Education (20091210110003). References [1] T. Akao, K. Kobayashi, M. Aburada, Enzymic studies on the animal and intestinal bacterial metabolism of geniposide, Biol. Phar. Bull. 17 (1994) 1573–1576.
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