1Department of Pharmacology, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
2YD Life Science Research Institutes, YD Life Science Co.Ltd., Seoul 139-743, Republic of Korea
3College of Pharmacy, Gachon University, Incheon, 406-799, Republic of Korea
Anxiety disorder, one of the most common psychiatric disorders affecting all age groups (Singh and Singh, 2002), encompasses several different forms of a type of mental illness characterized by abnormally and pathologically excessive fear and anxiety (Shin and Liberzon, 2010). The National Institute of Mental Health reported that anxiety disorders are a serious medical illness that affect approximately 19 million adults in the USA (Cryan and Holmes, 2005). It is difficult to overstate the fact that the rapidly increasing prevalence of anxiety disorders creates a burden on health systems around the world. Furthermore, anxiety disorder, classified as a mood disorder, not only causes significant disruption in psychological well-being, but also increases the risk of cardiovascular morbidity and mortality (Albert
Researchers have been searching for effective and safe agents with fewer side effects than existing anxiolytic agents, such as benzodiazepines, barbiturates, and antidepressants. Recently, many studies have focused on specific targets of brain neurotransmitter systems such as GABAA receptor and 5-HT1A receptor (Bailey and Toth, 2004; Clènet
The GABAergic system, the primary inhibitory neurotransmitter system, is thought to play an important role in anxiety disorders. Several pharmacologic agents, primarily with benzodiazepine structures, have been used to target the GABAergic system (Lydiard, 2003). GABAA receptors (ionotropic) and GABAB receptors (metabotropic) are widely expressed in the central nervous system. The GABAA receptor is known to be more associated with the acute stress response, and anxiolytic agents used clinically, such as benzodiazepines, target this receptor (Rudolph and Möhler, 2006). GABAA receptor agonists have shown anxiolytic-like effects in animal models of anxiety disorder (Rodgers and Dalvi, 1997), while a GABAA receptor antagonist, bicuculline, increased anxiety in rats tested by the widely-used elevated plus maze (EPM) (Miller
The brain serotonergic system, one of the well-characterized neurotransmitter systems related to emotion, is involved in mediating various behaviors included appetite, insomnia, depression, and anxiety disorders. The serotonergic system acts through at least 14 distinct receptor subtypes, exhibiting various complex physiological effects. Although the roles of individual receptor subtypes are not completely understood, many researchers have focused on the 5-HT1A receptor subtype in particular (Heisler
The aim of this study was to investigate the effects of
Male ICR mice (four-weeks-old, weighing 24-27 g) were purchased from DaeHan Biolink (Eumseong, Korea). Eight to ten animals were housed per cage. They were allowed access to water and food ad libitum, and maintained at constant temperature (23±1℃) and humidity (55±5%) under a 12 h light/ dark cycle (lights on 07:00-19:00 h) for one week before the experiments began. Mice were divided randomly into groups. All experiments were conducted in accordance with the NIH Guidelines on the Care and Use of Laboratory Animals, and our protocol was approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University.
(+)-Bicuculline and WAY 100635 (Sigma, St. Louis, MO, USA) were used in the EPM test. Thirty minutes before the CWE oral administration, (+)-bicuculline (0.3 and 1 mg/kg), WAY 100635 (0.3 and 1 mg/kg), or saline was administered intraperitoneally. Behavioral experiments were performed 1 h after the CWE administration. We chose dose and time point of (+)-bicuculline and WAY 100635 injentions based on the previous study (Yu
Samples were analyzed using the Agilent 1100 HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA), equipped with a quaternary solvent delivery system, an autosampler, and a DAD detector. Separations were carried out on a J’sphere ODS-H80 column (250×4.6 mm, 4 μm, YMC Co., Ltd. Japan). Chlorogenic acid was detected at 40℃ and 310 nm in isocratic elution mode using methanol (A) and 0.1% phosphoric acid in water (B). The elution profile was as follows: 0-20 min., 20% A in 80% B. Chlorogenic acid was prepared at 1 mg/ml and 10 μl was injected as an external standard. The CWE quantification assay was performed in triplicate.
The EPM test was performed according to the method described by Lister, with modifications (Lister, 1987). The standard plus maze consisted of two open arms (30×5 cm) and two closed arms with a wall (30×5×5 cm) connected to a central zone (5×5 cm) to form a cross. It was elevated to a height of 50 cm above the floor. A video camera was suspended above the maze to record the experiment. The maze floor and walls were constructed from opaque polyvinyl plastic, and the open arms had a low (0.5 cm) edge. The mouse was placed on the central zone facing an open arm. The maze floor was cleaned thoroughly between trials using 10% ethanol. The time spent in the open arm and number of open arm entries with four paws was recorded for a 5-min period using the video-based Ethovision 3.1 system. This test was performed under light (20 ± 5 lux) to encourage closed-arm entries. The parameters were calculated using the following formula: percentage of time spent in the open arm (%) = (time spent in the open arms/ sum of time spent in each arm)×100; percentage of open arm entries (%) = (number of entries in the open arms/total number of entries)×100.
All data are expressed as the mean ± SEM and were analyzed with Prism 5.0 software (Graphpad Software, Inc., San Diego, CA, USA). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Newman- Keuls multiple comparison test in order to detect inter-group differences. A
Chlorogenic acid, the functional component of CWE, was analyzed by HPLC. HPLC analysis of the standard substances showed that the retention time of chlorogenic acid was 11.44 min (Fig. 1). The chlorogenic acid content in CWE was determined from the liner regression equation of the calibration graph and was found to be 0.035%.
The control group mice typically avoided spending time in the open arms because of the height above the floor and the light. Fig. 2 shows that acute treatment with CWE (500 mg/kg, p.o.) markedly increased the time spent in the open arms compared with the vehicle group (F(3,21)=3.268,
As shown in Fig. 3, the administration of 500 mg/kg CWE significantly increased the time mice spent in the open arms compared with the vehicle group (p<0.05). This effect of CWE was blocked by pretreatment with the GABAA receptor antagonist, bicuculline, at 0.3 mg/kg and 1 mg/kg (F(3,41)=4.051,
The mice treated with CWE (500 mg/kg) were concurrently administered WAY 100635, a 5-HT1A receptor antagonist, in order to determine if the anxiolytic effects of CWE may involve the serotonergic neurotransmitter system, particularly the 5-HT1A receptors. As shown in Fig. 4, CWE (500 mg/kg) significantly increased the time spent in the open arms (F(3,40)=4.148,
The findings presented here support the idea that CWE has an anxiolytic-like effect through the GABAergic and se-
rotonergic systems. To investigate the anxiolytic-like effects of CWE in mice and to elucidate possible mechanisms, we used the EPM test with a GABAA receptor antagonist and a 5-HT1A receptor antagonist. In our study, CWE-administered mice showed a dose-dependent increase in the time spent in the open arm of the maze, an effect that was markedly blocked by a GABAA receptor antagonist and by a 5-HT1A receptor antagonist.
Anxiety models are grouped into two types involving either conditioned (potentiated startle) or unconditioned (social interaction and light/dark exploration tests) anxiety, and the EPM test is an excellent way to study unconditioned and spontaneous behavior (Rodgers and Dalvi, 1997). The EPM test, which may be the most popular model for measuring anxiety in animals, is based on the strong conflict between rodents’ proclivity toward dark, enclosed alleys (approach) and an unconditioned fear of brightly lit areas, heights or open spaces (avoidance) (Walf and Frye, 2007). In the EPM test of mice treated with CWE alone, CWE increased the time spent in the open arms in a dose-dependent manner. This finding suggests that CWE may alleviate unconditioned anxiety.
The GABAA receptor is known to be a mediator of unconditioned anxiety. GABAA receptors contain several subunits (α1-6, β1-3, γ1-3, δ, ε, π, ρ1-3, θ) encoded by 19 different genes (Luscher
The 5-HT1A receptor is also known as a modulator of unconditioned anxiety. Several studies have reported that 5-HT1A receptor antagonists have an anxiolytic effect in anxiety models (Rodgers and Cole, 1994; Griebel
However, the specific substance that causes the anxiolyticlike effects of CWE has not yet been identified. For this reason, we investigated the contents of CWE using quantitative HPLC analysis with chlorogenic acid as a standard compound. We found that CWE contains 0.035% chlorogenic acid, a functional ingredient. Several studies reported that chlorogenic acid has anticarcinogenic activity and anti-oxidative activity (Tanaka
In summary, our findings show that a single treatment with CWE confers anxiolytic effects in the mouse EPM test. These effects are blocked by both bicuculline and WAY 100635. Taken together, our results suggest that CWE has anxiolyticlike effects mediated by regulation of the GABAA and 5-HT1A receptors.