Abstract: Background: Semen Zizyphi Sponosae (SZS), a traditional Chinese medicine for the treatment of insomnia and anxiety symptoms. The present study aimed to discuss whether total alkaloids from SZS have anti-depression effect and investigate the antidepressant potential of total alkaloids isolated from Semen Zizyphi Sponosae (TAS) and its possible monoaminergic mechanism in mice. Methods: Mouse models of depression including the forced swimming test (FST), the Tail Suspension Test (TST) and Chronic Unpredicted Mild Stress (CUMS) were used to evaluate the effects of TAS. The possible mechanisms were examined by using fluorospectrophotometry to measure monoamine neurotransmitters in mice hippocampus and frontal cortex. The contents of monoamine neurotransmitters including 5-hydroxytryptamine (5-HT), Norepinephrine (NE) and Dopamine (DA). Results: The results showed that intragastric administration of the TAS (5, 10, 20 mg kg-1) or Venlafaxine 9.4 mg kg-1 significantly reduced the duration of immobility in TST and FST. However, the effect was not dose-dependent. TAS 10 and 20 mg kg-1 significantly increases the 5-HT levels in both of the two brain regions. Conclusion: These findings demonstrated that TAS produced an antidepressant-like effect and the mechanism of action involves the serotonergic, noradrenergic and monoamine oxidase enzyme systems although underlying mechanism still remains to be further elucidated.
INTRODUCTION
Depression is a chronic, recurring and potentially life-threatening illness that affects around 10-15% of the population across the globe. The World Health Organization predicts that depression will be the second most prevalent cause of illness-induced disability by the year 2020 (Murray and Lopez, 1997; Mathers and Loncar, 2006).
It is well known that monoamine neurotransmitters such as serotonin, noradrenaline and dopamine in the central nervous system play a key role in the pathophysiology of depression (Elhwuegi, 2004). The monoamine hypothesis predicts that the major neuro-chemical process in depression is the impairment of monoami-nergic functions associated with decreased levels of serotonin, noradrenaline and dopamine (Delgado, 2006). At present, several types of classical antidepressants are primary modulators of monoamine neurotransmitters. Most drugs for depression are poorly tolerated because of adverse side-effects, such as hypotension, arrhythmias, insomnia and sexual dysfunction (Wong and Licinio, 2001). Therefore, there is an urgent need for research and development of more effective antidepressant therapies without any or with less adverse effects. Nowadays, the use of traditional medicine such as traditional Chinese medicine, traditional Ayurvedic medicine has provided us a prospective alternative in the treatment of depression (Singh et al., 2009; Yi et al., 2009).
Semen Zizyphi Spinozae (SZS) is a very famous Chinese conventional herbal drug. It is used as an analgesic, tranquilizer and anticonvulsant in oriental countries such as Korea and China for over 2500 years to improve sleep quality and prolong sleep duration in patients with insomnia or anxiety (Mahajan and Chopda, 2009). Previous studies reveal that SZS contain many pharmacologically active components such as alkaloids, flavones and triterpenes (Lee et al., 1996). Sufficient researches suggest the major components of SZS (Guo et al., 1998) such as alkaloids (Ma et al., 2008), saponins (Cao et al., 2010) and flavonoids have hypnotic effects. A study indicated that the alkaloids and flavonoids of the seeds can produce central inhibitory activity (Park et al., 2004). However, very few researches are found to illustrate the antidepressant-like effect of TAS. Hence, in the present study, except for the FST and TST tests in mice, the Chronic Unpredicted Mild Stress (CUMS) model of depression in mice was used to further elucidate the antidepressant-like effects of TAS. In addition, the probable mechanism of antidepressant-like activity was explored by analyzing the monoamine neurotransmitter concentration.
MATERIALS AND METHODS
Plant material: Semen Zizyphi Spinozae (SZS) were purchased from a local herbal medicine market (Tianjin, China). The voucher specimen was deposited at the herbarium located in the College of Pharmacy, Tianjin Medical University (No. D060922) and authenticated by Professor Ye Zhou in Tianjin Medical University, based on their microscopic and macroscopic characteristics.
Preparation of total alkaloids: Alkaloids extract from Semen zizyphi spinozae was prepared as follows: SZS weighing about 200 g were homogenized to a fine powder and then macerated for 24 hx2 with 0.5% hydrochloric acid (1:7, w/v) in total. The two filtrates were collected and mixed together, centrifuged (2400 r min-1) for 10 min and the supernatant was chromatographed on a macroporous resin X-5 column (10x80 cm, Naikai Chemical Co., China) and eluted successively with distilled water, 70% ethanol, 95% ethanol. The 70% ethanol fraction was evaporated to a powder under vacuum at 40°C and further dissolved in distilled water. The solution was basified with NH4OH (pH 10) and extracted with ethyl acetate. The ethyl acetate extract were concentrated under reduced pressure at 40°C to obtain total alkaloids. The final yield was approximately 2.57% (w/w). The residue was detected by TLC. In TLC, the characteristic spots of including jujuboside A and B were detected and no spots of flavonoids and jujubosides were observed.
With anhydrous ethanol configured to TAS solution of 0.5 M. The content of total alkaloids was determined by UV. The equation of linear regression was Y = 0.0277X-0.0027 (r = 0.9997). The total alkaloids concentration was linear relationship with absorbance in the range of 6.6~33 23 μg mL-1. The average recovery was 100.0% and RSD was 1.61%. The content of TAS is 54.4%.
Reagents and chemicals: Venlafaxine (Wyeth Medica Ireland Kildare, Ireland) Reserpine (Bangmin pharmaceuticals Limited). All other reagents used in the study were of analytical grade.
Animals: Male ICR mice (weighing 18-20 g) purchased from Animal Center, Institute of Radiological Medical, Chinese Academy of Medical Sciences (SCKX-2009-0004). Each mouse was used for only one experiment. The animals were housed 10 per cage (440x270x178 mm) under controlled conditions of light (12 h light/dark cycle, lights on at 7:00 a.m.), temperature (22±2°C) and humidity 50-60% with free access to food and water. The animals were acclimatized to the laboratory for at least 3 days before they were tested. 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 Care and Use of Laboratory Animals.
Drug administration: The animals were randomized into control and experimental groups and divided into six groups (n=10 group). Animals in group 1 and 2 were administered with distilled water. Animals in groups 3, 4 and 5 were administered with the total alkaloids from Semen Zizyphi Spinozae at the doses of 5, 10 and 20 mg kg-1. Animals in group 6 were administered with venlafaxine at the doses of 9.4 mg kg-1 or fluoxetine at the doses of 7.5 mg kg-1. All drugs were orally administered the short-term (7 days) and sub-chronically (14 days) between 9:00-14:00 h. The behavioral tests were conducted 1 h after the sub-chronically (14 days) and last treatment, respectively.
Antidepressant activity evaluation
Tail suspension test (TST): The TST was performed according to the method
described by Steru et al. (1985). Briefly, mice
were individually suspended by tail with a clamp (1 cm from the tip of the end)
in box (25x25x30 cm) with the head 5 cm from the bottom. 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 to be immobile only when
they hung passively and completely motionless. Conventional antidepressants
decrease the immobility time in this test (Steru et al.,
1985).
Forced swimming test (FST): The FST is credited for having good predictive validity for detecting antidepressant activity, which was described and modified slightly by Porsolt et al. (1977). Mice were forced to swim in glass cylinders (20 cm heightx14 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 small movements necessary to keep its head above the water. The duration of immobility was scored during the last 4 min of the 6 min test period. Decrease in the duration of immobility was considered to be a measure of antidepressant-like effect (Gao et al., 2012).
Monoamine neurotransmitters assays: Animals receiving drugs for 14 days were used for determinations of NE, DA and 5-HT levels. Mice were sacrificed by decapitated and the total hippocampus and frontal cortex were rapidly dissected out on an ice-cooled dissection slab, weighed and stored at -80°C for the monoamine analysis.
The content of NE, DA and 5-HT were measured as described previously (Welch and Welch, 1969; Curzon and Green, 1970), with slight modifications.
Briefly, the brain tissue was homogenized in acidified n-butanol and shaken for 5 sec. Then, the suspension was centrifuged at 3000xg for 5 min at 4°C. The supernatant was mixed with n-heptane and 0.1 M HCl, shaken for 10 sec and centrifuged at 3000xg for 5 min at 4°C. The aqueous phase was used for the further analysis.
To determine NE and DA levels, 0.5 mL of the aqueous phase sample was pipetted into test tubes. 1.7 mL of phosphate buffer (pH7.2), 0.4mL of 0.1 M disodium ethylenediamine tetraacetic acid and 0.1 M iodine solution were added in order, then, let the tubes rest for 2 min after adding 0.5 mL of 0.2 M alkaline sodium sulfite, let them rest 2 min again. 0.5 mL of 6 M acetic acid was added at last. After heating in a boiling water bath for 2 min, tubes were cooled in water. Activation and fluorescent wavelengths of NE were at 284 and 480 nm, respectively. To determine DA, samples were heated for 2 min and cooled again. Activation and fluorescent wavelengths were at 320 and 369 nm, respectively (To detect the content of reagent blank and sample blank, it was just needed to put alkaline sodium sulfite before iodine solution).
To determine 5-HT, 0.4 mL of the aqueous phase sample was pipetted into test tubes and 0.1 mL of 1% o-phthalaldehyde in 10 M HCl and 0.1 mL of 0.02% sodium periodate were added successively. After heating in a boiling water bath for 10 min, the tubes were cooled in water and the fluorescence was measured. Activation and fluorescent wave were at 356 and 468 nm (Reagent blank was not added standard substance and 10 M HCl was used as a substitute for samples in sample blank).
Concentrations of NE, DA and 5-HT were calculated by compared with its respective standard substance. Obtained values were reported in ng g-1.
Statistical analysis: All data were expressed as Mean±S.E.M. Multiple group comparisons were performed using one-way Analysis of Variance (ANOVA) followed by Dunnett’s test in order to detect inter-group differences. A value of p<0.05 was considered statistically significantly for analysis.
RESULTS
Effects of TAS on the immobility time in the tail suspension test (TST): After one week treatment, the animals with TAS reduced the duration of immobility in the TST in a dose-dependent manner (27.5, 38.5% of reduction in the immobility time, compared with control group respectively), although the difference from the control at the lowest dose (5 mg kg-1) was not statistically significant shown in Fig. 1a. After the two-week treatment, TAS at 5, 10 and 20 mg kg-1 significantly decreased the immobility time in mice (17.0, 43.1, 46.6% of reduction in the immobility time, compared with control group, respectively) (p<0.001) shown in Fig. 1b. The experiment results showed that TAS required repeated administration to start showing its effects on immobility in the TST. The effect of TAS was similar to that of Venlafaxine (9.38 mg kg-1), used as a positive control.
Venlafaxine also induced a significant decrease when compared with the vehicle-treated group (p<0.001).
Effects of TAS on the immobility time in the forced swim test (FST): Figure 2a and b shows the effects of TAS for 7 and 14 days on the duration of immobility in the FST. As depicted in Day 14 shows at Fig. 2b, the TAS given by oral route at doses of 5, 10 and 20 mg kg-1 significantly decrease the immobility time in FST as compared to the control group. Venlafaxine induced effects of decreasing the immobility time in FST (p<0.001). The results showed that the FST is with the increase of dosage. So TAS required repeated administration to show its effects on immobility in the FST.
Effects of TAS on monoamine neurotransmitter levels in different brain regions
of mice
Hippocampus: Fig. 3(a-c), shows the
NE, DA and 5-HT levels in the hippocampus after two-week TAS treatment. It shows
after two-week treatment, the vehicle-treated group induced a significant decrease
in 5-HT (p<0.05, p<0.01, respectively) levels and no significant change
in NE and DA levels when compared with the control group. The 5-HT levels were
significantly increased only after the TAS extract treatment at the dose of
10 and 20 mg kg-1 (p<0.01, p<0.01, respectively) when compared
with the vehicle-treated group.
Frontal cortex: Fig. 4a-c shows the NE, DA and 5-HT levels in the frontal cortex after two-week TAS treatment. As shown in Fig. 4, after two-week treatment, the vehicle-treated group induced a significant decrease in 5-HT (p<0.05, p<0.01, respectively) levels and no significant change in NE and DA levels when compared with the control group. The 5-HT levels were significantly increased after the TAS extract treatments at the dose of 10 and 20 mg kg-1 (p<0.05, p<0.01, respectively) when compared with the vehicle-treated group.
DISCUSSION
It is suggested that behavioral study plays an important role in the evaluation of antidepressant drugs (Xu et al., 2008). In an attempt to mimic excessive human day-to-day stress, a number of animal tests are being used to investigate the mechanisms of depressive diseases and to screen potential new antidepressants. Tail Suspension Test (TST) (Steru et al., 1985) and Forced Swimming Test (FST) (Porsolt et al., 1977) are the most commonly predictive tests. Both of them reflect a depressive behaviour generally considered as despair or hopelessness when compared with control group. This two tests are thought to be highly predictable for antidepressant effects in humans (McArthur and Borsini, 2006). In the present study, we use the short-term and sub-chronic effects of TAS as the animal models, respectively. After the 7 days treatment TAS reduced the duration of immobility in the TST and FST. However, there was no statistical significance of immobility decrease induced by TAS (5, 10 and 20 mg kg-1) for 7 days treatment in the TST (p>0.05) was observed. After repeated treatment for 14 days, TAS decreased immobility time at all dosages used both in TST and FST. Likewise, the effect of TAS showed a dose-dependent manner, with a profile comparable to that observed for the classical antidepressant drug venlafaxine. The experiment results showed that TAS required repeated administration to start showing its effects on immobility in the TST.
So we speculated that sequence of mild stressors develop a series of abnormal the onset of antidepressant-like activity by TAS also required a chronic treatment in the mouse forced swim test.
CUMS model is based on the finding that rodents exposed to an unpredictable, relatively continuous behavioral and physiological responses that are reminiscent of those observed in depressed patients.
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). So in our study, we detected the brain monoamine level by Fluorescence Spectrophotometry after two-week treatment for CUMS-induced mice. We use the front and the hippocampus which involve in emotional, motivational and mnemonic processes, may be related to the expression of depression (Drevets, 2001).
Our results showed that TAS 10 and 20 mg kg-1 significantly increase the 5-HT levels in both of the two brain regions. The results suggest that TAS might exhibit its antidepressant-like effects via regulating serotonergic system.
CONCLUSION
In summary, total alkaloids from SZS exerts antidepressant-like effect in the FST and TST in mice. It is also presumed that the antidepressant-like activity of TAS may be related to the central monoaminergic neurotransmitter system, though other mechanisms remain possible.
ACKNOWLEDGEMENT This study was supported by the National Science Foundation of China (No. 81173530) and the Key Program of Tianjin Municipal Natural Science Foundation (No.12JCZDJC25900).