Available online at www.sciencedirect.com Life Sciences 82 (2008) 741–751 www.elsevier.com/locate/lifescie Antidepressant-like behavioral and neurochemical effects of the citrus-associated chemical apigenin ⁎ Li-Tao Yi, Jian-Mei Li, Yu-Cheng Li, Ying Pan, Qun Xu, Ling-Dong Kong State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, PR China Received 14 July 2007; accepted 16 January 2008 Abstract Apigenin is one type of bioflavonoid widely found in citrus fruits, which possesses a variety of pharmacological actions on the central nervous system. A previous study showed that acute intraperitoneal administration of apigenin had antidepressant-like effects in the forced swimming test (FST) in ddY mice. To better understand its pharmacological activity, we investigated the behavioral effects of chronic oral apigenin treatment in the FST in male ICR mice and male Wistar rats exposed to chronic mild stress (CMS). The effects of apigenin on central monoaminergic neurotransmitter systems, the hypothalamic–pituitary–adrenal (HPA) axis and platelet adenylyl cyclase activity were simultaneously examined in the CMS rats. Apigenin reduced immobility time in the mouse FST and reversed CMS-induced decrease in sucrose intake of rats. Apigenin also attenuated CMS-induced alterations in serotonin (5-HT), its metabolite 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA) levels and 5-HIAA/ 5-HT ratio in distinct rat brain regions. Moreover, apigenin reversed CMS-induced elevation in serum corticosterone concentrations and reduction in platelet adenylyl cyclase activity in rats. These results suggest that the antidepressant-like actions of oral apigenin treatment could be related to a combination of multiple biochemical effects, and might help to elucidate its mechanisms of action that are involved in normalization of stress- induced changes in brain monoamine levels, the HPA axis, and the platelet adenylyl cyclase activity. © 2008 Elsevier Inc. All rights reserved. Keywords: Apigenin; Antidepressant; Serotonin; 5-Hydroxyindoleacetic acid; Dopamine; Corticosterone; Adenylyl cyclase Introduction establishing normal mood by antidepressants (Duman et al., 1997; Nestler et al., 2002a; Coyle and Duman, 2003). Recent Depression is a serious emotional disorder with estimated research in the field has had the goal of discovering new targets lifetime prevalence as high as 21% of the general population in and developing novel therapeutics that act faster with higher some developed countries (Gainotti et al., 2001; Wong and efficacy and fewer side effects. Licinio, 2001; Nestler et al., 2002a,b). The neurobiology of Dysregulation of the hypothalamic–pituitary–adrenal (HPA) depression and its response to antidepressant treatment are axis is one of the most prominent neurobiological findings in not well understood. Some of the research on depression has major depressive disorder, and is considered as another im- focused on the interactions between the monoamine neuro- portant mechanism in the investigation of new antidepressant transmitters and their reuptake and receptor proteins. However, agents (Heuser et al., 1996; Nickel et al., 2003; Young et al., pharmacotherapy for depression often requires week- or month- 2004; Aihara et al., 2007). Adenylyl cyclase is an enzyme long treatments despite the fact that antidepressants immedi- that regulates the physiological effects of numerous drugs and ately affect the brain monoamine neurotransmission (Nestler, hormones through the production of cyclic adenosine-3′,5′- 1998), suggesting that other mechanisms may be involved in re- monophosphate (cAMP). Clinical and epidemiologic research has provided suggestive evidence regarding the association be- tween adenylyl cyclase activity and major depression (Cowburn ⁎ Corresponding author. Tel.: +86 25 8359 4691; fax: +86 25 8359 4691. et al., 1994; Reiach et al., 1999). Patients with major depression E-mail address: [email protected] (L.-D. Kong). were observed to have lower platelet adenylyl cyclase activity 0024-3205/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.01.007 742 L.-T. Yi et al. / Life Sciences 82 (2008) 741–751 (Cowburn et al., 1994; Menninger and Tabakoff, 1997). No- Furthermore, other reports demonstrated that apigenin inhibited ticeably, antidepressants could dramatically attenuate the reduc- γ-aminobutyric acid (GABA) receptor function and reduced tion of platelet adenylyl cyclase activity in depressed patients N-methyl-D-aspartate (NMDA) receptor function. Since dif- (Hines and Tabakoff, 2005). Thus, platelet adenylyl cyclase ferent GABA and NMDA receptor antagonists were effective activity might serve as a biological marker for major depression in many animal models of depression, these could account for and the therapeutic effect of antidepressants (Reiach et al., apigenin's antidepressant activity (Skolnick, 1999; Nakazawa 1999; Donati and Rasenick, 2003; Hines and Tabakoff, 2005; et al., 2003). These findings indicated that the antidepressant- Abdel-Razaq et al., 2007). like effects of apigenin might not be explained by only one Citrus fruits may be of potential interest to pharmaceuti- mechanism. To better understand the pharmacological activity of cal and food industries since they contain bioactive bioflavo- apigenin, we investigated the behavioral effects of oral apigenin noids with health-related properties. These components act as treatment in the FST in male ICR mice and in the chronic mild anti-oxidants in various biological systems (Morel et al., 1993; stress (CMS) model in male Wistar rats. The effects of apigenin Salah et al., 1995). They prevented pregnancy (Garg et al., on monoaminergic function in various brain regions, serum 2001), inhibited cancer cell proliferation (Manthey and Guthrie, corticosterone concentrations (an index of the HPA axis status), 2002), and displayed anti-allergic and anti-inflammatory ac- and platelet adenylyl cyclase activity were simultaneously studied tivities (Struckmann and Nicolaides, 1994). Early animal stud- in the CMS rats. ies confirmed that citrus fragrance reduced immobility time in the forced swimming test (FST) (Komori et al., 1995a,b) and Materials and methods accelerated the metabolic turnover of dopamine (DA) in hip- pocampus and of serotonin (5-HT) in prefrontal cortex and Materials striatum. Moreover, these effects were significantly blocked by pre-treatment with apomorphine, a nonselective DA receptor Apigenin was obtained from Shanxi Huike Botanical agonist, but not by agonists or antagonists to 5-HT receptor and Development Co., Ltd. (purityN98% by HPLC). Fluoxetine α-2 adrenaline receptor, indicating that citrus exerted anti- hydrochloride was purchased from Changzhou Siyao Pharma- depressant-like effects via modulating the 5-HT and DA func- ceuticals Co., Ltd. (P. R. China). All other chemicals used tions (Komiya et al., 2006). It was reported that citrus fragrance were of high-purity analytical grade obtained from commercial markedly reduced the doses of antidepressant treatment needed sources. for depressed patients in the clinic (Komori et al., 1995c). These observations have aroused our interest in searching for new Animals agents with antidepressant-like action from citrus. Apigenin (Fig. 1), one type of bioflavonoid widely found in Male ICR mice (Laboratory Animal Center, Nanjing Uni- citrus fruit, has been demonstrated to have anti-oxidation, anti- versity of Traditional Chinese Medicine, Jiangsu Province, P. R. inflammatory, and anti-tumor activities (Chen et al., 2005; Czyz China), weighing 23–25 g, were used. Animals were housed 5 et al., 2005; Fang et al., 2005; Hougee et al., 2005). Apigenin per cage (320×180×160 cm) under a 12-h/12-h light/dark was found to exert a variety of pharmacological actions on the schedule with the lights on at 07:00 a.m. and had free access to central nervous system, such as anxiolytic and sedative pro- tap water and food pellets. Ambient temperature and relative perties (Avallone et al., 2000; Zanoli et al., 2000). It was humidity were maintained at 22±2 °C and 55±5%, respectively. reported that acute intraperitoneal (i.p.) administration They were allowed at least 1 week to adapt to the laboratory of apigenin in ddY mice decreased immobility time in the environment before experiments. Experiments, performed by an FST and attenuated swim stress-induced decrease in DA turn- observer that was unaware of the treatment each mouse had over in amygdala and increase in DA turnover in hypothalamus, received, were carried out between 1:00 p.m. and 3:00 p.m. indicating that apigenin possessed antidepressant-like ef- Male Wistar rats (220–250 g), purchased from the Lab- fects, which might be mediated by dopaminergic mechanisms oratory Animal Center, Nanjing University of Traditional (Nakazawa et al., 2003). In addition, apigenin inhibited mono- Chinese Medicine, Jiangsu Province, P. R. China, were used in amine oxidase (MAO) activity (Lorenzo et al., 1996; Han et al., the experiments. Except as described below, the rats were sin- 2007). MAO inhibitors increase the levels of brain mono- gly housed and were kept on a 12-h light/dark cycle under con- amines, such as 5-HT, that have been related to the allevia- trolled temperature at 22±2 °C and humidity at 55±5%. They tion of clinical depression (Kanazawa, 1994; Wouters, 1998). were allowed free access to a laboratory chow diet and water. All studies were conducted in accordance with the Institu- tional Animal