Antidepressant-like effects of flavonoids extracted from Apocynum venetum leaves in mice : the involvement of monoaminergic system in mice

The present study investigated a possible antidepressant-like activity of flavonoids extracted from Apocynum venetum leaves using two predictive tests for antidepressant effect on rodents: the forced swimming test (FST) and the tail suspension test (TST). Additionally, the monoaminergic mechanisms involved in the antidepressant-like effect of AV-extract in the mouse forced swimming test (FST) were evaluated. The extract (25, 50 and 100 mg/kg, i.g.) produced antidepressant-like effects in the FST and TST, without accompanying changes in ambulation distances (open-field test). The antidepressant-like effects of AV-extract (50 mg/kg, i.g.) was prevented by the pretreatment of mice with ketanserin (5 mg/kg, s.c., a serotonin 5-HT2A receptor antagonist), cyproheptadine (3 mg/kg, i.g., a serotonin 5-HT2 receptor antagonist), prazosin (1 mg/kg, i.g., an α1-adrenoceptor antagonist), yohimbine (1 mg/kg, i.g., an α2-adrenoceptor antagonist) or propranolol (2 mg/kg, i.g., aβ-adrenoceptor antagonist), SCH23390 (0.05 mg/kg, i.g., a dopamine D1 receptor antagonist) and sulpiride (50 mg/kg, i.g., a dopamine D2 receptor antagonist). By contrast, pretreatment of mice with WAY 100635 (0.1 mg/kg, s.c., a serotonin 5HT1A receptor antagonist) did not counteract the antidepressant-like effect of AV-extract in the TST. It can be concluded that the AV-extract produces an antidepressant-like effects in the FST and in the TST through interaction with the serotonergic (5-HT2A and 5-HT2 receptors), noradrenergic (α1-adrenoceptor, α2-adrenoceptor, a β-adrenoceptor) and dopaminergic (D1 and D2 receptors) systems. Taken together, our results suggest that AV-extract deserves further investigation as a putative alternative therapeutic tool that could help the conventional pharmacotherapy of depression.


INTRODUCTION
Depression is a major psychiatric disorder affecting nearly 17% of the world population and imposes a substantial health burden on society (Nemeroff, 2007;Yu et al., 2002).According to the monoamine theory, which is a widely accepted explanation for depression, the major neurochemical process in depression is the impairment of monoaminergic functions and the decrease of serotonin, noradrenaline and dopamine levels (Delgado, 2000).Antidepressants increase the availability of these monoamines at the synapse, which may induce longer-term adaptive changes by modulating the monoaminergic functions and promoting neurogenesis (Elhwuegi, 2004;Dailly et al., 2004).Serotonergic, noradrenergic and dopaminergic systems represent the major targets of current therapeutic treatments and drug development.Yet, there are a few major issues with conventional antidepression drugs.(1) Low remission rate: Although they are mostly effective, only about 50% of individuals with depression showed full remission (Berton and Nestler, 2006).( 2) Low side effects: There are antidepressants that are not suitable for many patients because of the side effects.(3) Slow action: For many of these drugs, it takes several weeks to achieve their clinical efficacy.Hence, there is a growing interest in complementary and alternative medicine (CAM) among depression patients, with the general belief that "Natural is better" (Pilkington et al., 2006;Sarris and Kavanagh, 2009).Examples of plants with confirmed anti-depression effects include Akebiae quinata (Zhou et al., 2010), Albizzia julibrissin (Kim et al., 2007) and Bupleurum falcatum (Kwon et al., 2010).
Discovering new sources of natural products for depression treatment will continue to be an important field of research.Apocynum venetum L. (Apocynaceae) has shown a great promise for its antidepressant-like effects.A flavonoid extract from its leaves was found to markedly shorten the immobility time in a forced swimming test (FST), indicating antidepressant activities (Butterweck et al., 2001).The main flavonoids in A. venetum leaves are hyperoside and isoquercitrin (Butterweck et al., 2001).There is evidence that the A. venetum extract affects monoamine levels (Butterweck et al., 2003), we are interested in investigating the underlying mechanisms.Recently, we demonstrated that flavonoids from A. venetum leaves could generate a neuroprotective effect on corticosterone-induced neurotoxicity in PC12 cells (Zheng et al., 2011).
The purpose of the present study was to gain further insight into a possible involvement of noradrenergic, dopaminergic and serotonergic systems using laboratory mice.

Plant material and preparation of the flavonoids extracted from Apocynum venetum leaves
Dry Leaves of A. venetum leaves was purchased from Tong Ren Tang Co., Beijing.A sample of 10 g of leaves was extracted three times in a refluxed condenser for 1 h each with 200 ml 70% ethanol.The combined extract was evaporated until dryness, dissolved in 20 ml hot water, adjusted to pH 3.0 with sulfuric acid, and then filtered.The filtrate was chromatographed on a macroporous resin D101 column (10 × 80 cm, Naikai Chemical Co.China) and eluted sequentially with 100 ml water and 70% 100 ml ethanol.The aqueous ethanol fraction was evaporated to dryness to obtain A. venetum leaves extract.The extract contained 3.5% hyperoside and 3.7% isoquercitrin, respectively.High performance liquid chromatography (HPLC) analytical conditions were as follows: Column: SHISEIDO CAPCELL PAKC18 (UG) 4.6 mm i.d.3150 mm, detector at 330 nm, mobile phase: 0.1% TFA in water/0.1% TFA in acetonitrile 85: 15, flow rate: 1.0 ml/min.Equipment: Waters 2695 with a Waters 2998 DAD detector.Hyperoside appeared at 33.8 min and isoquercitrin at 39.7 min.

Animals
Male ICR mice weighing 18 to 22 g were purchased from the Experimental Animal Center, Changchun Institute of Biological Products, Jilin, China.Animals were under a normal 12 h/12 h light/dark schedule (with the lights on at 07:00 a.m).Ambient temperature and relative humidity were maintained at 22 ± 1°C and at 55 ± 5%, respectively.All mice had free access to tap water and food pellets, and were given a standard chow and water ad libitum for the duration of the study.November 14, 1988).All efforts were made to minimize animals suffering and to reduce the number of animals used in the experiments.

Drug treatments
Different groups of mice, 10 animals per group, were used for drug treatment and for each test.All drugs were administered by intragastric (i.g.) in a constant volume of 10 ml/kg body weight, except fluoxetine that were administered by oral (p.o.) gavage, and WAY 100635 and SCH23390 which were administered by subcutaneous route (s.c.).Appropriate vehicle treated groups were also assessed simultaneously.The animals were used only once in the test.

Experiment 1: To text the antidepressant-like effect of AVextract
Animals were divided into five experimental groups: one 0.9% saline control group, one fluoxetine group (5 mg/kg) and three AVextract treatment groups (25, 50, and 100 mg/kg).The administration volume was 20 ml/kg-body weight.To investigate its possible antidepressant-like effect, AV-extract was administrated by oral route 60 min before the forced swimming test (FST), tail suspension test (TST) or OFT.

Experiment 2: To assess the involvement of the serotonergic system in the antidepressant-like effect of AV-extract
To assess the involvement of the serotonergic system in the antidepressant-like effect of AV-extract in the TST, mice were pretreated with WAY 100635 (0.1 mg/kg, s.c., a serotonin5-HT 1A receptor antagonist), cyproheptadine (3 mg/kg, i.g., a serotonin 5-HT 2 receptor antagonist), ketanserin (5 mg/kg, i.g., a serotonin 5-HT 2A receptor antagonist) or vehicle.After 60 min, they received AV-extract (50 mg/kg, i.g.) or vehicle, and were tested in the TST 60 min later.

Experiment 3: To investigate the possible involvement of the noradrenergic system in the antidepressant-like effect of AVextract
To investigate the possible involvement of the noradrenergic system in the antidepressant-like effect of AV-extract in the TST, animals were pretreated with prazosin (1 mg/kg, i.g., an α 1adrenoceptor antagonist), yohimbine (1 mg/kg, i.g., an α 2adrenoceptor antagonist), propranolol (2 mg/kg, i.g., a βadrenoceptor antagonist) or vehicle.After 60 min they received AVextract (50 mg/kg, i.g.) or vehicle which were tested in the TST 60 min later.

Experiment 4: To test the possible involvement of the dopaminergic system in the antidepressant like effect of AVextract
To test the possible involvement of the dopaminergic system in the antidepressant like effect of AV-extract in the TST, animals were pretreated with SCH23390 (0.05 mg/kg, s.c., a dopamine D 1 receptor antagonist), sulpiride (50 mg/kg, i.g., a dopamine D 2 receptor antagonist) or vehicle.After 60 min they received AVextract (50 mg/kg, i.g.) or vehicle and were tested in the TST 60 min later.The doses of the drugs used above were determined according to literature data, which were reported not to increase the locomotor activity (Gay et al., 2010).

Forced swimming test
Mice were individually forced to swim in an open cylindrical container (diameter 10 cm, height 25 cm), containing 19 cm of water (depth) at 25 ± 1°C; the total amount of time each animal remained immobile during a 6-min session was recorded (in seconds) as immobility time, as described previously (Machado et al., 2009).Each mouse was judged to be immobile when it ceased struggling and remained floating motionless in the water, making only those movements necessary to keep its head above water.A decrease in the duration of immobility is indicative of an antidepressant-like effect (Porsolt et al., 1977).

Tail suspension test
The tail suspension test (TST) was conducted as previously described (Steru et al., 1985), with some modifications.Briefly, mice were individually suspended by the tail with a clamp (10 mm from the tail tip in a box (250 × 250 × 300 mm) with the head 50 mm from the bottom.Testing was carried out in a darkened room with minimal background noise.Each mouse was suspended for a total of 6 min, and the duration of immobility was recorded during the final 4 min interval of the test.Mice were considered immobile only when they hung passively and completely motionless.The test sessions were recorded by a video camera and scored by an observer Zheng et al. 767 blind to treatment.

Open-field test
To assess the effects of the extract from AV-extract on locomotor activity, the mice were individually housed in a rectangular container made of dark polyethylene (40 × 40 × 25 cm) in a dimly lit room equipped with a video camera above the center of the floor, as described previously (Kim et al., 2007) with slight modification, and locomotor activity was measured.The locomotor activity was monitored by a computerized video-tracking system using the S-MART program (Pan Lab, Barcelona, Spain).The animals were allowed to adapt for 1 h in the container, and the distance they traveled was recorded during the last 10 min of a total 20 min test.
The locomotor activity was measured in centimeters.The floor surface of each chamber was thoroughly cleaned with 70% ethanol between tests.

Statistical analyses
All data were expressed as mean ± standard error of mean (SEM).
To compare experimental and control groups, we used one-or twoway ANOVA, followed by post-hoc Dunnett's test using the SPSS software (SPSS Inc., Chicago, USA).A value of P < 0.05 was considered statistically significant for analysis.The figures were obtained by the Statistical Analysis System (Graph Pad Prism 4, Graph Pad Software, Inc., San Diego, CA).

Effects of AV-extract on the immobility time in the FST and TST and on the locomotor activity in the OFT
AV-extract or the conventional antidepressant fluoxetine given by oral route significantly decreased the immobility time in the FST [F (4, 45) = 16.25,P < 0.001] and TST [F (4, 45) = 22.37, P < 0.001].Administration of AV-extract (20, 50,100 mg/kg) and fluoxetine (5 mg/kg) resulted in a significant decrease in the immobility time.As shown by the OFT, AV-extract (25, 50 and 100 mg/kg) did not lead to significant changes in ambulation distance (2212.6 ± 4 8.6, 2253 ± 89.9, and 2275.2 ± 98.9 mm, respectively) relative to the vehicle group (2297.3± 100 mm).This indicates a lack of apparent association of immobility in the tests with changes in locomotor activity.

Involvement of the serotonergic system
The result presented in Figure 2A showed that the pretreatment of animals with ketanserin (5 mg/kg, i.g., a 5-HT2A receptor antagonist) was able to prevent the antiimmobility effect of AV-extract (50 mg/kg, i.g.) in the TST.

Involvement of the noradrenergic system
The result presented in Figure 3A showed that the pretreatment of animals with prazosin (1 mg/kg, i.p., an α1-adrenoceptor antagonist) was able to inhibit the antiimmobility effect of AV-extract (50 mg/kg, i.g.) in the TST.

DISCUSSION
Behavioral study is an important approach in evaluating anti-depressant drugs, and forced swimming test (FST) and tail suspension test (TST) are popular tools.The characteristic behavior scored in these tests, termed "immobility", is an indicator of the degrees of severity of behavioral despair; the lower are the scores, the less severe is the despair.Antidepressant drugs are able to reduce the immobility time in rodents (Porsolt et al., 1977).The antidepressant-like effect of A. venetum was first demonstrated by Butterweck et al. (2001) using a forced swimming test (FST) in rats, and the present study has provided more detailed behavioral data using mice.
Our results showed that administration at 100, 50 and 25 mg/kg (i.g.) for 10 days significantly reduced immobility time in FST (P < 0.001, p < 0.05 and P < 0.01, respectively; Figure 1A).Administration at 100, 50 mg/kg (i.g.) also significantly reduced immobility time in TST (P < 0.01 and P < 0.001, respectively; Figure 1B).In line with these observations, treatments with AV-extract at 100, 50 and 25 mg/kg did not cause significant changes in amulation distance (2212.6 ± 48.6, 2253 ± 89.9 and 2275.2 ± 98.9 mm, respectively) compared to the vehicle group (2297.3± 100 mm; Figure 1C).These effects were comparable to those found with fluoexetine treatment.Taken together, AV-extract possesses a clear antidepressant-like effect in all animal models used.
It is generally accepted that improving brain monoaminergic functions is effective in treating depression, and the serotonergic, noradrenergic or dopaminergic systems have become the targets for development of antidepressants (Lambert et al., 2000;Esposito, 2006).The serotonergic system has long been implicated in the pathogenesis of anxiety and depression (Heninger et al., 1996).Some of the most compelling evidence involves the  alleviation of depression caused by serotonin selective reuptake inhibitors (SSRIs), which increase the availability of serotonin at the synapse (Malagie et al., 2002).The serotonergic system is a neuromodulatory system interacting with other neurotransmissions and participating in the elaboration of an adapted response of the central nervous system to external stimuli (Grimaldi et al., 1999).Depressions are often associated with a down regulation of 5-HT 1A receptors in the hippocampus and in the temporal lobe (Gross et al., 2000).5-HT 2 receptors, especially 5-HT 2A and 5-HT 2C subtypes, have been shown to be involved in neurochemical changes mediated by antidepressants (Cryan and Lucki, 2000;Elhwuegi, 2004).Preclinical reports showed that the preferential 5-HT 2A receptor agonist DOI enhances the antidepressant-like effect of some compounds (Zomkowski et al., 2004) in the mouse FST.Accordingly, antidepressant desipramine treatment decreased 5HT 2 receptors densities in the rat brain (Goodnough and Bake, 1992).In the present study, the pretreatment with pretreatment with 5-HT 2 receptor antagonist cyproheptadine and 5-HT 2A receptor antagonist ketanserin prevent the anti-immobility effect induced by AV-extract in the TST, whereas WAY 100635 was ineffective in reversing the immobility time in the TST; these results are indicative of participation of 5-HT 2 and 5-HT 2A receptors, but not 5-HT 1A receptor, in the antidepressant-like effect of AV-extract in the mouse TST (Figure 2).
Noradrenergic system is another target for antidepressants (Maj et al., 2000).Increased levels of α 1adrenoceptor were found in the prefrontal cortex of depressed individuals (García-Sevilla et al., 1999).A down regulation of a 2 -adrenergic receptors was documented in depression (Brunello et al., 2003).There is an upregulation of β-adrenoceptor in depressed patients and a down-regulation after chronic antidepressant treatment in mice (Leonard et al., 1997).In our study, pretreatment of mice with prazosin and yohimbine or propranolol was able to reverse the antidepressant-like effect of the AVextract, suggesting participation of α 1 -, α 2 -and αβadrenoceptors in the antidepressant-like effect of the extract in mouse (Figure 3).
The relationship between dopaminergic system and depression was confirmed by the fact that antidepressants act on the dopaminergic system (Klimek et al., 2002).Common symptoms of depression such as anhedonia, dysphoria and avolition are likely caused by a functional deficit of dopaminergic transmission (Heinz et al., 1994).Many antidepressant drugs such as SSRIs, imipramine (a tricyclic antidepressant) and bupropion (an atypical antidepressant that inhibits the reuptake of dopamine) act activating dopamine D 1 and D 2 receptors (Renard et al., 2001;Yamada et al., 2004).In our experiments, the pretreatment with SCH23390 or sulpiride inhibited the anti-immobility effect of AV-extract.Thus, our results suggested a participation of both dopamine D 1 and D 2 receptors in the antidepressant-like effects of AVextract.
In summary, our results provided pharmacological and biochemical evidence for the previously reported antidepressant-like effects of AV-extract in the FST (Butterweck et al, 2001).We have further shown that these effects are apparently related to noradrenergic (α 1adrenoceptor, α 2 -adrenoceptor, aβ-adrenoceptor), dopaminergic (D 1 and D 2 receptors) and serotonergic (5-HT 2A and 5-HT 2 receptors).

Figure 1 .
Figure 1.Effects of AV-extract and fluoxetine on immobility time in the mouse FST (A), TST (B), and OFT (C).The immobility time was measured in mice receiving 0.9% saline (vehicle), 25, 50 and 100 mg/kg AV-extract, 5 mg/kg fluoxetine for 10 days.The number of mice in each group was 10.Data were expressed as mean ± S.E.M. *P < 0.05 and **P < 0.01 and ***P < 0.001 vs FST or TST or OFT with vehicle group.
All procedures were performed in accordance with the published guidelines of the China Council on Animal Care (Regulations for the Administration of Affairs Concerning Experimental Animals, approved by the State Council on October 31, 1988 and promulgated by Decree No. 2 of the State Science and Technology Commission on