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Antidepressant-like effect of the water extract of the fixed combination of Gardenia jasminoides, Citrus aurantium and Magnolia officinalis in a rat model of chronic unpredictable mild stress.


Background: Water extract of the fixed combination of Gardenia jasminoides Ellis fruit, Citrus aurantium L fruit and Magnolia officinalis Rehd, et Wils, bark, traditional name--Zhi-Zi-Hou-Po (ZZHPD) is used for treatment of depressive-like symptoms in traditional Chinese medicine for centuries.

Hypothesis/Purpose: The present study aimed to explore antidepressant-like effects and potential mechanisms of ZZHPD in a rat model of chronic unpredictable mild stress (CUMS).

Study design: Antidepressant-like effects of ZZHPD were investigated through behavioral tests, and potential mechanism was assessed by neuroendocrine system, neurotrophin and hippocampal neurogenesis.

Methods: Antidepressant-like effects of ZZHPD (3.66, 7.32 and 14.64 g/kg/day) were estimated through coat state test, sucrose preference test, forced swimming test and open-field test. Effects of ZZHPD on hypothalamic-pituitary-adrenal (HPA) axis were evaluated by hormones measurement and dexamethasone suppression test. In addition, the expression of brain-derived neurotrophic factor (BDNF) in hippocampus was measured, as well as hippocampal neurogenesis was investigated by doublecortin (DCX) and 5-bromo-2-deoxyuridine/neuronal nuclei (BrdU/NeuN).

Results: The results demonstrated that ZZHPD significantly reversed the depressive-like behaviors, normalized the levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT), restored the negative feedback loop of HPA axis and improved the levels of BDNF, DCX and BrdU/NeuN compared with those in CUMS-induced rats.

Conclusion: The above results revealed that ZZHPD exerted antidepressant-like effects possibly by normalizing HPA axis function, increasing expression of BDNF in hippocampus and promoting hippocampal neurogenesis.


Water extract of the fixed combination of Gardenia jasminoides. Citrus aurantium and Magnolia officinalis


Chronic unpredictable mild stress

Hippocampal neurogenesis

Hypothalamic-pituitary-adrenal axis


Depression, a complex psychological illness which can be triggered by chronic psychosocial stress in vulnerable humans (Keller et al. 2007) and characterized by loss of pleasure, mood disturbance and suicidal tendencies (Tanti and Belzung 2010), gives rise to a significant increase in prevalence and produces a considerable socioeconomic burden in the fast-paced lifestyles with high-pressure populations worldwide.

The hypothalamic-pituitary-adrenal (HPA) axis is one of complicated neurobiological mechanisms for development of depression. The overproduction of glucocorticoid hormones reflected dysfunction or hyperactivity of HPA axis in depressive disorders, which provided significant indicator of depression in response to stressors (de Kloet et al. 2005). In addition, depression was reported to be associated with stress-induced reduction in neurotrophin levels in hippocampus and hippocampal neurogenesis, leading to hippocampal atrophy (Schmidt and Duman 2007). However, antidepressants could restore these symptoms. Hence, normalization of HPA axis system, elevation of neurotrophin levels and restoration of hippocampal neurogenesis as prime mechanisms of antidepressant actions appeared to be special interests.

Water extract of the fixed combination of Gardenia jasminoides Ellis fruit, Citrus aurantium L. fruit and Magnolia officinalis Rehd. et Wils. bark, traditional name--Zhi-Zi-Hou-Po (ZZHPD) is used in traditional medicinal system in China. It was originally recorded in "Shang Han Lun" written by Zhang Zhongjing in Eastern Han Dynasty of ancient China. As a classical prescription, it has been reported that ZZHPD successfully improved depressive-like behaviors in mice (Yao et al. 2013). However, the underlying pharmacological mechanisms of ZZHPD on HPA axis, neurotrophin levels and hippocampal neurogenesis remained to be ambiguous.

In the present study, the antidepressant-like effects of ZZHPD on CUMS-induced rats were evaluated through behavioral tests. Furthermore, the potential mechanism was explored through whether ZZHPD could restore dysfunction of HPA axis, improve expression of BDNF in hippocampus and increase hippocampal neurogenesis.

Materials and methods


Male Sprague-Dawley rats weighing 220-250 g were supplied by the Experimental Animal Center of Shenyang Pharmaceutical University. Rats were maintained on a standard light/dark cycle under controlled temperature (22 [+ or -] 2 [degrees]C) and humidity (50 [+ or -] 10%) with certified standard diet and water ad libitum. The animals were allowed to acclimatize for 3 weeks with training for sucrose preference test before the experiment. The animal study was carried out in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of China. All efforts were made to minimize suffering.

Materials and reagents

Gardenia jasminoides Ellis fruit (batch number: 1205002), Citrus aurantium L. fruit (batch number: 1205001) and Magnolia officinalis Rehd. et Wils. bark (batch number: 120901) were purchased from Tong Ren Tang Chinese Medicine Co., Ltd. (Beijing, China) and authenticated by Professor Jincai Lu (School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, China) according to Chinese Pharmacopoeia (The Pharmacopoeia Commission of PRC, 2010). Voucher specimens were deposited in the Herbarium of Shenyang Pharmaceutical University. Fluoxetine (FLU), as a positive control, was obtained from Lilly S. A. (Alcobendas, Spain). Geniposide, hesperidin, neohesperidin, magnolol and honokiol were supplied by National Institutes for Food and Drug Control (Shenyang, China). Genipin-1-[beta]-gentiobioside was purchased from Shanghai Winherb Medical Technology Co., Ltd. (Shanghai, China). Solvents used as eluents were distilled water and HPLC grade acetonitrile from Fisher Scientific (Fair Lawn, NJ, USA).

Preparation of ZZHPD and quality analysis

The general preparation procedure of ZZHPD lyophilized powder is as follows. Gardenia jasminoides Ellis fruit, Citrus aurantium L. fruit and Magnolia officinalis Rehd. et Wils. Bark dry herbs were crushed into pieces and mixed in the ratio of 1:1:7 (w/w). The mixture was macerated in distilled water (1:10, w/v) for 0.5 h, then boiled for 1 h, subsequently the extraction was filtered through five layer gauzes, afterwards the residue was boiled again twice in a total volume of 8 and 5 times (w/v) weight of the herbs, respectively. After that, the supernatants was combined and concentrated under reduced pressure. Freeze-drying was used to produce lyophilized powder, and the drug extract ratio (DER genuine) was 5.5:1 (100%: 18.02%). It was in accordance with the European Medical Agency (EMA) guidelines. 14.7 g of extract (as dry extract) was equivalent to 81.4 g the fixed combination of herbal substances from Gardenia jasminoides Ellis, fruit (Gardeniae fructus)/Citrus aurantium L, fruit (Aurantii fructus immaturus)/Magnolia officinalis Rehd. et Wils., Bark (Magnoliae officinalis cortex) (1/1/7). The quality was controlled based on a three-dimensional high performance liquid chromatography (3D-HPLC). Typical chromatogram and three-dimensional fingerprint of ZZHPD were shown in Fig. 1.

The thin-layer chromatography (TLC) identification of single herbal extract was carried out according to Chinese Pharmacopoeia. Upon comparison with the standard solutions, special spots showed the same color tone and [R.sub.f] value. In addition, the amounts of main active markers were determined by HPLC to secure the stable contents according to Chinese Pharmacopoeia and some reports (Donato et al. 2014; Xu et al. 2008). The detailed quality analysis procedure and 3D-HPLC profiles of single herb were shown in Fig. SI from supplementary materials.

All the analytes showed good linearity ([r.sup.2] > 0.999), and the relative standard deviation (RSD%) of precision and repeatability were all <3.0%. The recoveries were in the range of 95-105%.

1 g of ZZHPD lyophilized powder contained geniposide (1.00 mg) and genipin-1-[beta]-gentiobioside (4.73 mg) both from Gardenia jasminoides Ellis fruit, hesperidin (24.20 mg) and neohesperidin (0.19 mg) both from Citrus aurantium L. fruit, magnolol (4.33 mg) and honokiol (4.23 mg) both from Magnolia officinalis Rehd. et Wils. bark.

Drug administration and treatment

The rats were divided into six groups based on sucrose preference results: control (CON) group, CUMS model group, FLU group (15 mg/kg), ZZHPD (3.66, 7.32 and 14.64 g/kg equivalent to the fixed combination of herbal substances) groups, and each group included 12 rats. The dosages of ZZHPD were decided based on the fact that ZZHPD was prescribed at a daily dose of 81.4 g of the fixed combination of herbal substances (Yao et al. 2013). We converted human dose into animal dose in accordance with body surface area principle and designed 7.32 g/kg as clinical equivalent dose (70 kg of Human and 0.2 kg of rat at a conversion factor of 0.018: Doserats = 81.4 * 0.018/0.2 [approximately equal to] 7.32 g/kg). All rats were first exposed to CUMS for 2 weeks followed by administering drugs daily for 3 weeks with the continued CUMS procedure. The CON group and CUMS group were given distilled water daily. The drugs were administered to rats in a dose volume of 10 ml/kg by gastric gavages once daily 30 min before stress exposure during the last 3 weeks. The experimental procedure was shown in Fig. 2.

Chronic unpredictable mild stress (CUMS) procedure

The CUMS procedure was carried out as previous report (Nollet et al. 2013) with minor modification. Briefly, the control rats were group-housed, and the CUMS-induced rats were isolated in individual cages. The CUMS-induced rats were randomly exposed to various stressors for 5 weeks: white noises for 24 h (alternative periods of 60 dBA noise for 10 min and 10 min of silence), wet bedding for 24 h (200 ml of water per individual cage to make the bedding wet), 24 h of food deprivation, 5 min of tail pinch (1 cm from the end of the tail), 23 h of water deprivation followed by exposure to empty water bottles for 1 h, stroboscope (120 flashes per minute) for 24 h, cold swim in 15 [degrees]C for 5 min, restraint stress for 6 h and light on for 24 h. The control rats were left undisturbed, and the CUMS-induced rats received randomly one of these stressors per day. The same stressor was not applied continuously for 2 consecutive days so that animals could not predict the occurrence of stimulation. The detailed protocol was shown in Table SI.

Coat state test

The coat state was recorded and evaluated in the head, neck, dorsal regions, and on the paws and tail as an indirect measure of grooming at the end of experiment. For each body area, a score of 0 was given for a well-groomed coat and 1 for an unkempt coat. The total score of coat state resulted from the sum of scores from five different body parts. The higher score showed that the coat state was in poor condition. The index has been pharmacologically validated in previous studies (Ducottet and Belzung 2004).

Sucrose preference test

The sucrose preference test was conducted as described previously with minor modifications (Willner et al. 1987). Briefly, rats were trained to adapt sucrose solution (1%, w/v) before the test. After adaptation, the rats were deprived of water for 24 h, followed by sucrose preference test, in which rats were housed in individual cage and had free access to two bottles with 100 ml of sucrose solution (1%, w/v) and 100 ml of water, respectively. The bottle position was balanced and switched regularly to avoid side preference. After 1 h, the amount of water and sucrose solution consumed was measured by weight, and the sucrose preference was calculated as a ratio of the amount of sucrose solution to that of total solution:

Sucrose preference (%) = sucrose consumption/ (water consumption + sucrose consumption)

Forced swimming test

The test was conducted according to the method of Porsolt et al. (Porsolt et al. 1978). Briefly, rats were placed in a plexiglass cylinder separately (60 cm tall, 30 cm diameter) filled to a depth of 25 [+ or -] 5 cm with 24 [+ or -] 1 [degrees]C water. Two swimming sections were conducted: an initial 15 min pretest, followed by a 6 min test 24 h later. The duration of immobility was measured during the last 4 min by observers blind to the treatment groups of rats. Duration of immobility was regarded as the time spent by rat on floating in the water without struggling and making only those movements necessary to keep its head above the water.

Open-field test

The open-field test was carried out as described previously (Monteggia et al. 2007). The open-field apparatus consisted of a square (100 cm x 100 cm x 40 cm) arena. The rat was placed individually into the corner of the open field and allowed to explore freely. The activity was tested during 6 mins in open field. A video tracking system (Ethovision, Noldus Systems, Wageningen, The Netherlands) was used to record the time spent in the arena, along with a named 'periphery zone', a named 'non-periphery zone' and a named 'center zone'.

Plasma hormone assay

The ACTH concentrations were measured using the adrenocorticotropic hormone ELISA kits (Nanjing Jiancheng Bioengineering Institute, China), and the CORT concentrations were measured using a corticosterone ELISA kits (Nanjingjiancheng Bioengineering Institute, China), following the manufacturer's instruction.

Dexamethasone suppression test

The dexamethasone suppression test was conducted to assess the effects of CUMS and ZZHPD treatments on the HPA axis negative feedback. The rats were injected intraperitoneally with the glucocorticoid receptor agonist dexamethasone (0.1 mg/kg in 0.9% NaCl, D4902, Sigma-Aldrich) or saline (0.9% NaCl). After 30 min, the rats were subjected to an acute stressor (forced swimming) for 5 min. Then, 120 min after dexamethasone injection, blood samples were collected and centrifuged (5 min, 4000 r/min, 4 [degrees]C) for hormone assay, and supernatants were stored at -80 [degrees]C until analysis.

After blood collection, the rats were fully anesthetized with sodium pentobarbital (35 mg/kg, i.p.), and then transcardially perfused with saline solution through ascending aorta followed by 4% paraformaldehyde dissolved in 0.1 M phosphate buffer saline (PBS) (pH = 7.4). The brains were removed, post-fixed for 12 h at 4 [degrees]C in the same fixative and transferred to 30% sucrose solution for 24 h. Then, coronal sections (25 [micro]m) were cut on a freezing microtome (AS-620, Shandon, Astmoor, UK), which were stored at -80 [degrees]C until further processing.


The sections were removed from -80 [degrees]C to adapt to room temperature, and then rinsed three times with 0.01 M PBS for 5 min consecutively. Antigen retrieval was soaked in 10 mM trisodium citrate buffer (pH = 6.0, 85-95 [degrees]C) for 5 min and washed with PBS for thrice. The sections were steeped in 0.3% hydrogen peroxide in methanol for 20 min and blocked with normal goat serum for 1 h at 37 [degrees]C, followed by incubation with rabbit polyclonal anti-BDNF antibody (1:500, Abeam: ab101747) or rabbit polyclonal anti-DCX antibody (1:800, Abeam: ab18723) overnight. Subsequently, sections were incubated with secondary antibody (biotinylated goat-anti rabbit) for 1 h and visualized with DAB (ZSGB-BIO, Beijing, China). Finally, sections were dehydrated in graded alcohol solutions, cleared in xylene and covered slips with mounting medium. The Olympus BX40 microscope (Olympus, Tokyo, Japan) with Image-Pro Plus 6.0 was used for capturing the image. The sum of integrated optical density (IOD) of BDNF and the number of DCX across the hippocampus were measured in each section.


The Immunofluorescence was conducted with both BrdU and NeuN as previously described (Nollet et al. 2012). The sections were incubated in HCl (2 M) for 20 min at 37 [degrees]C to fracture the DNA structure of the labeled cells, and then rinsed twice in 0.1 M borate buffer (pH = 8.5), blocked with 10% normal goat serum (diluted in 0.3% Triton X-100) for 1 h at 37 [degrees]C followed by incubating with rabbit monoclonal anti-BrdU antibody (1:50, Abeam: ab6326) and mouse polyclonal anti-NeuN antibody (1:1000, Millipore: mab377) diluted in 0.1 M PBS overnight at 4 [degrees]C. Secondary antibodies consisted of Cy3-conjugated goat anti-rabbit IgG (H + L) (1:200, Beyotime Institute of Biotechnology) and AlexaFluor 488-conjugated goat anti-mouse IgG (H + L) (1:200, Beyotime Institute of Biotechnology) was used. Sections were incubated for 1 h at 37 [degrees]C and covered slips with anti-fade mounting medium (Pro-Long Gold, Molecular Probes).


Data were expressed as mean [+ or -] standard error of mean (SEM). The data were performed by one-way ANOVA followed by Fisher's LSD test in our study. Statistical analysis was carried out using SPSS 19.0 software for Windows (SPSS Inc, Chicago, IL, USA). A value of P < 0.05 was considered statistically significant.


Effect of ZZHPD on the coat state test in CUMS rats

As shown in Fig. 3A, CUMS protocol gave rise to a significant deterioration of the coat state compared with the CON group at the end of experiment (P <0.001). ZZHPD (3.66,7.32 and 14.64 g/kg) and FLU (15 mg/kg) treatments counteracted markedly the CUMS-induced deterioration of coat state after 3 weeks of treatments (P < 0.001).

Effect of ZZHPD on the sucrose preference in CUMS rats

The results of sucrose preference test were exhibited in Fig. 3B. There was no significant difference between the CON and CUMS group at the beginning of experiment (not shown). The percentage of sucrose preference was significantly reduced by CUMS (P < 0.01) at the end of experiment. ZZHPD at doses of 7.32 (P < 0.01), 14.64 g/kg (P < 0.05) and FLU at dose of 15 mg/kg (P < 0.05) showed a significant increase compared with the CUMS group.

Effect of ZZHPD on the forced swimming test in CUMS rats

As shown in Fig. 3C, CUMS significantly increased the immobility time in the forced swimming test compared to the CON group (P < 0.05). ZZHPD (7.32, 14.64 g/kg) and FLU (15 mg/kg) significantly decreased the duration of immobility time compared to the CUMS group (P < 0.01, P < 0.05, P < 0.05, respectively).

Effect of ZZHPD on the open-field test in CUMS rats

Fig. 3D exhibited the representative traces of rats movement in open-field test. CUMS lead to a significant reduction in the duration of center (Fig. 3E, P < 0.05) and non-periphery zone (Fig. 3F, P < 0.01), and increase in the duration of periphery zone compared with the CON group (Fig. 3G, P < 0.01). ZZHPD (7.32, 14.64 g/kg) and FLU (15 mg/kg) significantly reversed the duration in the center, periphery and non-periphery zone (P < 0.05 or P < 0.01).

Effects of ZZHPD on plasma hormone levels

As shown in Fig. 4A, CUMS procedure induced a significant increase in the plasma ACTH levels compared with the CON group (P < 0.01). After 21 days of treatment, ZZHPD (7.32, 14.64 g/kg) and FLU (15 mg/kg) significantly decreased plasma ACTH levels compared with the CUMS group (each P < 0.05).

Similarly, plasma CORT levels were strikingly increased by CUMS (P < 0.001). ZZHPD (7.32 and 14.64 g/kg) significantly reduced the plasma CORT levels (P < 0.05 and P < 0.01), and the FLU group also produced a marked reduction compared to the CUMS group (Fig. 4B, P < 0.01).

Effects of ZZHPD on HPA axis negative feedback

Dexamethasone suppression test showed that CUMS produced a significant reduction in the percentage of dexamethasone induced plasma corticosterone suppression (Fig. 4C, P < 0.05), which indicated the integrity of negative feedback of HPA axis was disrupted in CUMS group. However, the reduction could be reversed after chronic ZZHPD (7.32 and 14.64 g/kg) and FLU (15 mg/kg) treatments (P < 0.05 or P < 0.01).

Effects of ZZHPD on BDNF, DCX and BrdU/NeuN levels in the hippocampus in CUMS rats

The effects of ZZHPD and FLU on BDNF, DCX and BrdU/NeuN levels in hippocampus in CUMS-induced rats were exhibited in Fig. 5. The results revealed that CUMS induced a significant reduction in the expression of BDNF (Fig. 5A and B), DCX (Fig. 5C and 5D) and BrdU/NeuN (Fig. 5E and 5F) positive cells compared with the CON group (P < 0.01, P < 0.05, P < 0.01, respectively). ZZHPD (7.32, 14.64 g/kg) and FLU (15 mg/kg) treatments significantly increased the BDNF, DCX and BrdU/NeuN levels compared with the CUMS group (P < 0.05 or P < 0.01).


Up to date, it has been reported that genipin, one of important bioactive ingredients from Gardeniae Fructus, significantly reversed CUMS-induced behavioral changes, and potential mechanism involved in the dysfunctional regulation of BDNF levels in hippocampus (Wang et al. 2014). Previous study reported that Fructus Aurantii significantly decreased the immobility time in forced swimming test and increased the locomotor activity in open-field test (Zhang et al. 2012). Hesperidin, as a main active component of Fructus Aurantii, exhibited antidepressant-like effect in mouse tail suspension test, which was possibly mediated by increasing BDNF levels in hippocampus (Donato et al. 2014). Treatment with the mixture of honokiol and magnolol as the main components from Magnoliae Officinalis Cortex significantly decreased immobility time in mouse forced swimming test and tail suspension test, and reversed CMS-induced reduction of sucrose preference, of which antidepressant mechanism involved restoration of plasma CORT and normalization of HPA axis (Xu et al. 2008). In a word, the ZZHPD formula is rational, and its antidepressant mechanism need to be further explored.

The well-validated CUMS model, in which rats mimicked various behavioral and physiological symptoms that resemble those observed in depressive patients, has been used widely to study the pathophysiology of depression and evaluate antidepressant-like effect of diverse drugs (Willner 1997). Therefore, CUMS model was used to assess the antidepressant-like effects of ZZHPD in this study. Deterioration of coat state was one of the symptoms of depressed patients, and it was the most prevalent, reliable and well-validated measure used in the CUMS model of depression (Ducottet and Belzung 2005). In the present study, CUMS induced a significant deterioration of coat state. This was in line with previous studies that CUMS could induce depression-related physical changes (de Kloet et al. 2005; Nollet et al. 2012), and ZZHPD could significantly reverse deterioration of coat state. Besides, the sucrose preference index was used as parameter of anhedonia, which was a core symptom of depression. In the present study, CUMS significantly reduced sucrose preference index, which indicated decreased responsiveness to rewards, and this phenomenon could be restored by ZZHPD. It was well known that forced swimming test is a behavioral despair model to reflect recurrent thoughts of death, which was another important symptom of depression in humans (Porsolt et al. 1978). In accordance with previous findings, CUMS could significantly increase the immobility time in the forced swimming test, and this phenomenon was reversed by ZZHPD. Anxiety and depression have high lifetime prevalence and become the most common psychiatric disorders (Trivedi et al. 2006), so depression-related anxious behaviors were investigated in open field test. Open field test was based on the fact that rats feared to enter an open or bright arena to reflect emotionality, and the time spent in different spaces as crucial index was used to assess anxiety-like behavior. The results showed that CUMS significantly increased escape behavior in center zone and non-periphery zone, but opposite in periphery zone. It was noteworthy that ZZHPD could improve the anxious-like behaviors. Taken together, all above results suggested that ZZHPD exerted antidepressant-like effect in the CUMS-induced depression rats.

It is widely accepted that stress plays an important role in the development of depression. Considerable evidences suggested that long chronic stress exposure could give rise to disorder of HPA axis function and hypersecretion of corticosteroids, which has been considered to be closely associated with the pathogenesis of depressive disorder in humans Qozuka et al. 2003). High levels of ACTH and CORT were commonly observed in depressed patients and accompanied with the impairment of negative feedback of HPA axis (Pariante and Lightman 2008). In the present study, ZZHPD could normalize CUMS-induced high levels of ACTH and CORT. In addition, dexamethasone suppression test was used to assess the effect of ZZHPD on integrity of HPA axis negative feedback loop. Dexamethasone, as glucocorticoid receptor agonist, initiates negative feedback loop of HPA axis to reduce levels of ACTH and CORT. CUMS significantly decreased dexamethasone-induced plasma CORT suppression, which indicated that CUMS disrupted negative feedback regulation of HPA axis, while ZZHPD could restore negative feedback control of HPA axis. These results were also in line with the previous studies that antidepressant therapy could restore HPA axis function (Pariante and Lightman 2008). All above results suggested that ZZHPD might exert antidepressant-like effects by reduction of ACTH and CORT levels and restoration of negative feedback loop of HPA axis.

BDNF, as one of neurotrophins, supports the survival of central nervous system and plays a key role in pathophysiology of depression. Hyperactivity of HPA axis and excessive stress hormone induced by chronic stress could reduce expression of BDNF, which possibly had an impact on neuronal atrophy and cell loss, including regulation of hippocampal neurogenesis (de Kloet et al. 2005). A particular attention was focused on BNDF and hippocampal neurogenesis based on the fact that antidepressants could exert antidepressant-like effect through increasing BDNF expression in hippocampus, as well as BDNF was an important regulator of adult hippocampal neurogenesis (Lee et al. 2002). In addition, blocking of hippocampal neurogenesis could prevent part behavioral effects of antidepressants in rodents (Taliaz et al. 2010). Therefore, potential mechanisms of ZZHPD on anti-depression were assessed by BNDF and neurogenesis in hippocampus. In the present study, CUMS significantly reduced levels of BDNF, DCX and BrdU/NeuN in hippocampus. These results were consistent with previous studies that CUMS inhibited proliferation of hippocampal progenitor cells and destroyed function of neurogenesis (Nollet et al. 2012; Wang et al. 2014). Nevertheless, ZZHPD reversed CUMS-induced reductions in expression of BDNF, DCX and BrdU/NeuN in hippocampus, which suggested that ZZHPD might enhance BDNF and hippocampal neurogenesis to resist depression. Therefore, it was speculated that ZZHPD might exert antidepressant-like effects by increasing BDNF expression and promoting neurogenesis in hippocampus. However, the exact targets of antidepressant-like effects of ZZHPD remain unknown, and further research is needed.


In summary, the data presented in the study showed that ZZHPD significantly improved CUMS-induced depressive-like behaviors. Moreover, ZZHPD might exert antidepressant-like effects by normalizing HPA axis function, increasing expression of BDNF in hippocampus and promoting hippocampal neurogenesis to protect neuron from damage in response to chronic stress. These findings indicate that ZZHPD will be a promising candidate for relief from depression and facilitate its clinical usage.


Article history:

Received 10 June 2015

Revised 11 September 2015

Accepted 15 September 2015

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.


We thank Zhang Kuo for his pharmacological advice.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2015.09.004.


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Hang Xing (a), Kuo Zhang (b), Ruowen Zhang (c), Huiyan Shi (a), Kaishun Bi (a), Xiaohui Chen (a),*

(a) School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China

(b) Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China

(c) Stem Cell Institute, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Shelby Interdisciplinary Biomedical Building, Room 777 1825 University Blvd, Birmingham, AL 35294-0024, USA

Abbreviations: ZZHPD, traditional name--Zhi-Zi-Hou-Po; CUMS, chronic unpredictable mild stress; BDNF, brain-derived neurotrophic factor; DCX, doublecortin; ACTH, adrenocorticotropic hormone; BrdU/NeuN, 5-bromo-2-deoxyuridine/neuronal nuclei; HPA, hypothalamic-pituitary-adrenal; FLU, fluoxetine; CON, control; 3D-HPLC, three-dimensional high performance liquid chromatography; CORT, corticosterone; HPLC-DAD, high performance liquid chromatography-diode array detector.

* Corresponding author. Tel: +86 24 23986259; fax: +86 24 23986259.

E-mail address: (X. Chen).
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Author:Xing, Hang; Zhang, Kuo; Zhang, Ruowen; Shi, Huiyan; Bi, Kaishun; Chen, Xiaohui
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Dec 1, 2015
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