Antidepressant-Like Activity of the Ethanolic Extract from Uncaria lanosa Wallich var. appendiculata Ridsd in the Forced Swimming Test and in the Tail Suspension Test in Mice

This study investigated the antidepressant activity of ethanolic extract of U. lanosa Wallich var. appendiculata Ridsd (ULEtOH) for two-weeks administrations by using FST and TST on mice. In order to understand the probable mechanism of antidepressant-like activity of ULEtOH in FST and TST, the researchers measured the levels of monoamines and monoamine oxidase activities in mice brain, and combined the antidepressant drugs (fluoxetine, imipramine, maprotiline, clorgyline, bupropion and ketanserin). Lastly, the researchers analyzed the content of RHY in the ULEtOH. The results showed that ULEtOH exhibited antidepressant-like activity in FST and TST in mice. ULEtOH increased the levels of 5-HT and 5-HIAA in cortex, striatum, hippocampus, and hypothalamus, the levels of NE and MHPG in cortex and hippocampus, the level of NE in striatum, and the level of DOPAC in striatum. Two-week injection of IMI, CLO, FLU and KET enhanced the antidepressant-like activity of ULEtOH. ULEtOH inhibited the activity of MAO-A. The amount of RHY in ULEtOH was 17.12 mg/g extract. Our findings support the view that ULEtOH exerts antidepressant-like activity. The antidepressant-like mechanism of ULEtOH may be related to the increase in monoamines levels in the hippocampus, cortex, striatum, and hypothalamus of mice.


Introduction
Depression, a widespread incapacitating psychiatric ailment, imposes a substantial health burden on society [1]. Affective disorder are characterized by a disturbance of mood associated with alteration in behavior, energy, appetite, sleep, and weight [2]. According to the most accepted hypothesis of depression, the monoamine theory, patients with major depression have symptoms that are reflected changes in brain monoamine neurotransmitters, specifically norepinephrine (NE) and serotonin (5-HT) [3]. Clinical data suggests that dopamine (DA) is also involved in the pathophysiology and treatment of depression [4]. Medications such as tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), monoamine oxidase inhibitors (MAOIs), specific serotonin-norepinephrine reuptake inhibitors (SNRIs), 5-HT 2 receptor antagonists, and other heterocyclics are clinically employed for drug therapy [5]. However, these drugs can impose a variety of side-effects including sedation, apathy, fatigue, sleep disturbance, cognitive impairment, and sexual dysfunction, and so forth. Hence, there remains a pressing need for new effective and better-tolerated antidepressants.
Herbal therapies may be effective alternatives in the treatment of depression, such as Hypericum perforatum L. [6], Cordyceps sinensis [7], and Perilla frutescens [8]. The Uncaria species recorded in Chinese Pharmacopoeia and Taiwan Herbal Pharmacopoeia include Uncaria rhynchophylla 2 Evidence-Based Complementary and Alternative Medicine (Miquel) Jacks (abbrev. as UR), U. macrophylla Wallich. U. hirsuta Haviland (UH), U. sinensis (Oliver) Havil, and U. sessilifructus Roxburgh [9,10]. According to Flora of Taiwan, there are three different species of Gouteng in Taiwan: UR, UH, and U. lanosa Wallich var. appendiculata Ridsd (UL) [11]. However, UL is not recorded in Pharmacopoeia. In traditional Chinese medicine, Gouteng is categorized as a herb to extinguish wind, arrest convulsions, clear heat, and pacify the liver [12]. Gouteng is mainly used to treat cardiovascular and central nervous system ailments, including light headedness, convulsions, numbness, and hypertension [12]. Several studies demonstrate that the herb extract mainly acts on neuroprotective effect used to treat antiepileptic [13][14][15], anti-Parkinsonian [16], anti-Alzheimer's disease [17,18], anxiolytic [19], protective action against ischemia-induced neuronal damage [20,21], anti-inflammation [22]. Alkaloids are the active pharmacological component in Gouteng and comprise components include RHY, isorhynchophylline, hirsutine, hirsuteine, corynantheine, isocorynoxeine. RHY exhibited a similar pharmacological activity when compared with Gouteng [12]. RHY is an important active component of alkaloids separated from gambir plant (Gouteng in Chinese), RHY exerts the protective action primarily by inhibiting of NMDA and 5-HT 2 receptor-mediated neurotoxicity during ischemia [21]. RHY also affects the levels of serotonin in cortex, striatum, hippocampus, and hypothalamus [23,24]. From the above perspectives, we inferred that RHY is the key component of antidepressantlike activity of Gouteng. Gouteng possesses neuroprotective effect, regulation of monoamine transporters, macrophage theory [25], and regulation of glutamatergic system [26]. Our preliminary test indicated that ethanolic extract of U. lanosa Wallich var. appendiculata Ridsd. (UL EtOH ) contained the largest amount of RHY among Uncaria species in Taiwan. However, the antidepressant-like activity of UL EtOH has not been investigated, which encouraged us to investigate the effects of UL EtOH on depression problems.
In the present study, we aimed to investigate the effect of UL EtOH in FST and TST in mice. The behavioral despair tasks have good predictive value for antidepressant potency in humans [27]. Moreover, we investigated whether the effect of UL EtOH in FST and TST is dependent on its interaction with the 5-HT, NE, and DA receptors, and the brain monoamine neurotransmitter concentration. MAO activity was also tested by neurochemical and biochemical assays to confirm the participation of monoamine transmitters in treatment involving UL EtOH .

Animals.
Male ICR albino mice (weighing around 22 g), purchased from BioLASCO Taiwan Co., Ltd., were used in the present study. They were maintained at 22±1 • C with free access to water and food, under a 12 : 12 h light/dark cycle (lights on at 08:00 h). All manipulations were carried out between 9:00 and 15:00 h, with each animal used only once. All procedures in this study were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Committee on Animal Research, China Medical University. The minimum number of animals and duration of observations required to obtain consistent data were used.

Preparation of Plant
Extract. Dried 1 kg of UR, UH, and UL, made from the stems and hooks of plants, were sliced into small pieces and ground into a powder, and extracted four times with 5% ammonia solution and 70% ethanol. The extracts were filtered, combined, and concentrated under reduced pressure at 40 • C to obtain the UR EtOH , UH EtOH , UL EtOH extracts. The yield ratios of the UR EtOH , UH EtOH , UL EtOH extracts (120 g, 97 g, 115 g) were 12%, 9.7%, 11.5%.

HPLC Analysis of UR
EtOH , UH EtOH , and UL EtOH . The HPLC system consisted of a Shimadzu (Kyoto, Japan) LC-10ATvp liquid chromatograph equipped with a DGU-14A degasser, an FCV-10ALvp low-pressure gradient flow control valve, an SIL-10ADvp autoinjector, an SPD-M10Avp diode array detector, and an SCL-10Avp system controller. Peak areas were calculated using Shimadzu Class-LC10 software (Version 6.12 sp5). The column was a Phenomenex Synergi 4 Fusion-RP 80A column (250 mm × 4.6 mm). The gradient mobile phase was methanol (solvent A) and 0.01 mol/L triethylamine, and adjusted to adjust pH to 7.5 with glacial acetic acid (solvent B) solvent A : B = 60 : 40. The sample was Evidence-Based Complementary and Alternative Medicine 3 injected of 10 μL. The following gradient profile was run at 1.0 mL/min over 60 min. Peaks were detected at 274 nm with SPD-M10AVP (Shimadzu) detector. The peaks of UR EtOH , UH EtOH , and UL EtOH samples were identified by comparison with the standard solutions (RHY). The UR EtOH , UH EtOH , and UL EtOH solutions were quantified by spiking with a known amount of standard and also by comparing the area under curve. The repeatability of the method was evaluated by injecting the solution of UR EtOH , UH EtOH , and UL EtOH and standard solution three times, and the relative standard deviation (RSD) percentage was calculated.

Forced Swimming Test (FST).
The method was carried out on mice according to the method of Porsolt et al. [28]. Mice were placed in an open cylindrical container (diameter 10 cm, height 25 cm), containing 15 cm of water at 25 ± 1 • C. The duration of observed immobility was recorded during the last 4 min of the 6-minute testing period [29,30]. Mice are forced to swim in a restricted space from which they cannot escape and are induced to a characteristic behavior of immobility. 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. Decrease in the duration of immobility during the FST was taken as a measure of antidepressant activity.

Tail Suspension Test (TST).
The total duration of immobility induced by tail suspension was measured according to the method of Steru et al. [31]. Mice both acoustically and visually isolated were suspended 50 cm above the floor by adhesive tape placed approximately 1 cm from the tip of the tail. The time during which mice remained immobile was quantified during a test period of 6 min. Mice were considered immobile only when they hung passively and completely motionless.
To assess the effects of UL EtOH, on locomotor activity, mice were evaluated in the open-field paradigm as previously described [32]. Animals were individually placed in a box (40 × 60 × 50 cm). The mice were placed in the center and their behavior was noted immediately and continued for 5 min. The parameters such as resting time, total movement distance, total movement time, total movement were recorded by video camera and registered in the computer. During the interval of the test the apparatus was cleaned.

Pharmacological Treatments.
We investigated whether the antidepressant-like activity of UL EtOH in FST and TST is dependent on its interaction with IMI (a tricyclic antidepressant), MAP (a selective NE reuptake inhibitor), FLU (a selective 5-HT reuptake inhibitors), BUP (a selective DA reuptake inhibitor), CLO (a selective MAO-A inhibitor), and KET (a preferential 5-HT 2A receptor antagonist). To this end, mice were pretreated with UL EtOH (0.5 g/kg for two weeks' administration) or saline. They received IMI, FLU, KET, CLO (5 mg/kg for two weeks' administration), MAP (20 mg/kg for two weeks' administration), or BUP (4 mg/kg for two weeks' administration) 30 mins before being tested in FST and TST.
The doses of the drugs which do not affect locomotor activity and immobility time were selected on the basis of literature data [33][34][35] and our preliminary test.

Determination of Monoamines and Their Metabolites Levels in the Mice Frontal Cortex, Striatum, Hippocampus, and
Hypothalamus. Animals were divided into six groups (n = 6/group): control (0.9% saline), control versus FST, the three experiment groups (0.125, 0.25, 0.5 g/kg, for two weeks' administration) , and IMI (10 mg/kg for two weeks' administration).
Monoamines were measured according to the method of Renard et al. [36]. Briefly, mice were killed by cervical dislocation without anesthesia just after the FST. The brain was removed after a rapid dissection of frontal cortex, striatum, hippocampus, and hypothalamus were isolated. The four brain tissues were weighed and placed separately in 5 mL of ice-cold homogenizing solution (8.8 mg of ascorbic acid and 122 mg of EDTA in 1000 mL of perchloric acid 0.1 M). After homogenization, the solution was centrifuged at 10,000 ×g for 10 min at 4 • C. Twenty microliters of the resultant supernatant was injected in the high-performance liquid chromatography (HPLC) system. The levels of monoamines (NE, DA and 5-HT) and their metabolites (MHPG, DOPAC, 5-HIAA) were measured by HPLC (Waters 610) with electrochemical detection in the three brain tissues. The mobile phase [4.2 g/L] citric acid monohydrate, 6.8 g/L sodium acetate trihydrate, 0.8 g/L octanesulfonic acid sodium salt, 0.05 g/L tetrasodium ethylenediamine tetraacetate, 0.02% (v/v) dibutyl amine, and 7% (v/v) methyl alcohol) was delivered at 1.0 mL/min. The reverse-phase column used was a Merk Lichrospher 100 RP-18 endcapped column with a length of 12.5 cm and an internal diameter of 4.0 mm (E. Merk 50734). The compounds were measured at +0.75 V using a Bioanalytical Systems LC-4C electrochemical detector.
Mice were sacrificed and the brain tissues was rapidly frozen (−80 • C) until analyzed. The brain tissues was each homogenized in 50 mM phosphate buffer (pH 7.4) containing 0.5 mM EDTA and 0.25 M sucrose and stored at −80 • C. Protein content of the homogenate was determined using the method of Lowry et al. [37]. Mouse brain monoamine oxidase activity was measured following the method of Zhou and Panchuk-Voloshina [38]. Briefly, For the measurement of each type of MAO, serotonin was used as a substrate for MAO-A and benzylamine for MAO-B. The experiments were conducted at room temperature for 60 min in a reaction mixture with brain homogenates at a final protein concentration of 8 mg/mL. For the sensitivity assay, the brain homogenates with different protein concentrations were incubated in a reaction mixture of 200 mM Amplex Red, 1 mM benzylamine, and 1 U/mL HRP at room temperature for 60 min.
2.11. Statistical Analysis. All results are expressed as mean ± SEM. Data were analyzed by one-way ANOVA followed by Bonferroni , s multiple range test. The criterion for statistical significance was P < 0.05. All statistical analyses were carried out by using SPSS for Windows (SPSS Inc.).

HPLC Analysis of UR EtOH , UH EtOH , and UL
EtOH . The HPLC chromatogram shows that RHY is the major components among organic molecules of UR EtOH , UH EtOH , and UL EtOH . As shown in Figure 1, the content of RHY in UR EtOH and UL EtOH were 3.87 mg/g and 17.12 mg/g. UH EtOH did not detect the content of RHY.

Effect of Repeated Treatment with UL EtOH on the Immobility Time Both in the FST and TST.
In order to investigate whether UL EtOH can produce chronic changes in depressionrelated behavior in FST and TST, we treated mice with different dosages to mice via continuous oral administration for 14 days. UL EtOH decreased significantly the immobility time in FST (dose range: 0.0625-0.5 g/kg, p.o.; Figure 2). UL EtOH also caused a reduction in the immobility time in TST (dose range: 0.0625-0.5 g/kg, p.o.; Figure 3). In both tests, IMI at doses of 10 mg/kg produced a reduction of the immobility time that was stronger than that afforded by UL EtOH (Figure 3).

Effect of Repeated Treatment with UL EtOH on the Locomotor Activity in Mice.
In order to determine whether UL EtOH actually possesses an antidepressant-like activity, we tested the locomotion counts to exclude the excitatory or inhibitory effects after administration of UL EtOH . UL EtOH did not affect locomotor activity at the same doses that significantly reduced immobility response in the FST and TST (Figure 4).

Effect of Combination of UL EtOH with IMI, FLU, CLO, MAP, BUP, and KET on Immobility Periods in FST and TST.
The results depicted in Figure 5 show the effect of treatment of mice with IMI (5 mg/kg for two weeks' administration, a dose that did not affect the immobility time) on the reduction in immobility time elicited by UL EtOH (0.5 g/kg, p.o.). Post-hoc analyses indicated that the treatment of mice with IMI augmented the antidepressant-like activity of UL EtOH in FST and TST. The results depicted in Figure 6 show the effect of treatment of mice with FLU (5 mg/kg for two weeks' administration, a dose that did not affect the immobility time) on the reduction in immobility time elicited by UL EtOH (0.5 g/kg, p.o.). Post-hoc analyses indicated that the treatment of mice with FLU augmented the antidepressantlike activity of UL EtOH in FST and TST.
The results depicted in Figure 7 show the effect of treatment of mice with CLO (5 mg/kg for two weeks' administration, a dose that did not affect the immobility time) on the reduction in immobility time elicited by UL EtOH (0.5 g/kg, p.o.). Post-hoc analyses indicated that the treatment of mice with CLO augmented the antidepressantlike activity of UL EtOH in FST and TST.
The results depicted in Figure 8 show the effect of treatment of mice with MAP (20 mg/kg, for two weeks' administration, a dose that did not affect the immobility time) on the reduction in immobility time elicited by UL EtOH (0.5 g/kg, p.o.). Post-hoc analyses indicated that the treatment of mice with MAP did not augment the antidepressant-like activity of UL EtOH in FST and TST. The results depicted in Figure 9 show the effect of treatment of mice with BUP (4 mg/kg, for two weeks' administration, a dose that did not affect the immobility time) on the reduction in immobility time elicited by UL EtOH (0.5 g/kg, p.o.). Post-hoc analyses indicated that the treatment of mice with BUP did not augment the antidepressant-like activity of UL EtOH in FST and TST.
The results depicted in Figure 10 show the effect of treatment of mice with KET (5 mg/kg for two weeks' administration, a dose that did not affect the immobility    and hippocampus, the level of NE in striatum, and level of DOPAC in striatum. Table 5 summarizes the effect of UL EtOH and clorgyline on the activities of type A and type B monoamine oxidase in mouse brain. UL EtOH (0.5 g/kg) and clorgyline (10 mg/kg) inhibited the activity of type A monoamine oxidase in the mouse brain.

Discussion
In the present study, we analyzed the RHY content of Gouteng grown in Taiwan and choose the UL which has higher amount of RHY as the research sample. To raise the yield ratio of alkaloid, the researcher alkalized the three species of Gouteng by 5% ammonia solution, turning alkaloid salts into free alkaloid, followed by 70% ethanol extracting. After the above procedure, the UR EtOH , UH EtOH , and UL EtOH were produced. Afterwards, the researcher used HPLC method to analyse the RHY content of the samples. The analytical result of UL EtOH contained most RHY among all. However, from the study of Jung et al. [19], the aqueous extract of UR (UR DDW ) possesses anxiolytic activity by inhibiting WAY1005635 (the compounds that could selective block 5-HT 1A presynaptic receptors and prevent the negative feedback might be effective) [34]. In this study, the researcher analysed the RHY content of UR DDW . Result showed that UR DDW did not detect RHY (unpublished data) and suggested that the antianxiety activity of UR DDW was not related to RHY. The forced swimming and tail suspension tests are behavioral despair tests useful for probing the pathological mechanism of depression and for the evaluation of antidepressant drugs [39]. These tests are sensitive to all major classes of antidepressant drugs including tricyclics, serotonin reuptake inhibitors, monoamine oxidase inhibitors, and atypical [28]. Characteristic behavior scored in both tests is termed immobility, reflecting behavioral despair as seen in human depression [31]. The results presented here show, to our knowledge for the first time, that UL EtOH given orally is effective in producing significant antidepressant-like activity, when assessed in FST and in TST. The antidepressant-like activity of UL EtOH in FST and TST was not comparable but weaker than that of IMI, used as a standard antidepressant in a dose of 10 mg/kg. In FST and TST, psychostimulants are also shown to reduce immobility but in contrast to antidepressants they cause a marked motor stimulation. Locomotor activity test was also observed after UL EtOH treatment. We employed an additional locomotor activity test to check the motor stimulating activity of UL EtOH after tests. These results suggested that UL EtOH , at the same doses that produce an antidepressant-like activity, did not show significant locomotor stimulation. The antidepressant-like activity of UL EtOH is specific.
The precise mechanisms by which UL EtOH produced antidepressant-like activity are not completely understood. However, according to our results, the antidepressant-like activity of UL EtOH was additive to the treatment of animals with IMI (a-NE/5-HT reuptake inhibitor), FLU (a selective 5-HT reuptake inhibitor), CLO (a selective MAO-A inhibitor), and KET (a preferential 5-HT 2A receptor antagonist) when tested in FST and TST. This effect was not accompanied by hyperlocomotion (data not shown) that could produce a false-positive antidepressant-like activity. These suggest that UL EtOH might produce antidepressant-like activity by interaction with monoamines receptors, and monoamine oxidase, thereby increasing the levels NE, 5-HT, and DA in the brains of mice and was related to downregulation of 5-HT 2A receptor (inhibition of 5-HT 2A receptor expression exerts antidepressant-like activity) [40]. Moreover, this study suggests that the combination of UL EtOH with these antidepressants might be helpful in the treatment of depression.
Intensive research into the neurobiology of depression suggests that an increase in the monoamine levels at the synapse is believed to be the first step in a complex cascade of events that results in antidepressant activity [41]. Four brain regions were studied: the frontal cortex, the striatum, the hippocampus, and the hypothalamus, which are involved Integrating in important behavioral functions, such as emotion, motivation, and learning and memory [41,42]. Abnormal monoamine levels in four brain regions may be relevant to the depressed state. Our results show that UL EtOH increased the levels of 5-HT and 5-HIAA in cortex, striatum, hippocampus, and hypothalamus, the levels of NE and MHPG in cortex and hippocampus, the level of NE in striatum, and the level of DOPAC in striatum. The HPLC assay showed that a significant increase in DOPAC in the striatum was observed after UL EtOH treatment. The results from behavior and HPLC assay were inconsistent possibly because behavioral changes are not significantly sensitive to small changes in dopamine level in the brain. Integrating the HPLC, and pharmacological treatments results, we inferred that the anti-depression mechanism of UL EtOH might be partly due to its influence on the function of 5-HT,NE systems through the regulation of serotonergic and adrenergic receptors and/or the metabolism of 5-HT and NE. Values were the mean ± SEM (n = 6). # P < 0.05, ## P < 0.01, ### P < 0.001 as compared with the normal group. * P < 0.05, * * P < 0.01, * * * P < 0.001 as compared with the control versus FST group (one-way ANOVA following by Bonferroni's test). Value were the mean ± SEM (n = 6). # P < 0.05 as compared with the normal group. * P < 0.05, * * P < 0.01, * * * P < 0.001 as compared with the control group (one-way ANOVA following by Bonferroni's test). Value were the mean ± SEM (n = 6). # P < 0.05, ## P < 0.01, ### P < 0.001 as compared with the normal group. * P < 0.05, * * P < 0.01, * * * P < 0.001 as compared with the control group (one-way ANOVA following by Bonferroni's test). Value were the means ± SEM (n = 6). # P < 0.05, ## P < 0.01, ### P < 0.001 as compared with the Normal group. * * P < 0.01, * * * P < 0.001 as compared with the control group (one-way ANOVA following by Bonferroni's test). Value were the mean ± SEM (n = 6). * * P < 0.01 as compared with the control group (one-way ANOVA following by Bonferroni's test).
MAO exists in two subtypes, A and B. The original MAOIs are nonselective, inhibiting both forms. The A form of MAO preferentially metabolizes 5-HT and NE, the monoamines most closely linked to depression. The B form preferentially metabolizes trace amines, including phenethylamine. MAO-A and MAO-B metabolize DA and tyramine [43]. Hou et al. [44] concluded that the (+) catechin and (−) epicatechin of methanol extract of UR had inhibitory effect on MAO-B activity. However, due to the different extracting methods in this research, we did not detect the contents of (+) catechin and (−) epicatechin in UL EtOH. (unpublished data). Furthermore, based on the same studies have reported a positive correlation between oxidative stress and depression [45], and Gouteng has antioxidant activity [46]. We applied two-weeks oral administration of UL EtOH to conduct monoamine oxidase activity test. The results of our study reveal that UL EtOH inhibited MAO-A activity.
Several studies demonstrated that the herb extract and its active component RHY protect neurons against the ischemia, glutamate-, or dopamine-induced damage or death [20,21,47], and regulation of monoamine transporters [23,24]. From the above studies, we inferred that RHY might be the main active component in Gouteng's antidepression activity. Further studies are needed to verify the antidepressant activity of RHY and underlying mechanisms.
In conclusion, UL EtOH contained most RHY among Uncaria species of Gouteng in Taiwan. UL EtOH showed antidepressant-like activity in FST and TST. The mechanism of anti-depressive-like activity of UL EtOH was mediated by increasing the monoamines level, particularly 5-HT and NE in different brain regions of mice. Furthermore, UL EtOH was proofed to inhibit the activity of MAO A . From the present study, we conclude that UL EtOH is a worth developing Taiwanese specific medicinal plant, and thus we suggest that it should be included in Pharmacopoeia.