Journal of Ethnopharmacology 309 (2023) 116302

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Antidepressive mechanisms of rhynchophylline in mice with chronic

unpredictable stress-induced depression
En-Yu Liu a, 1, Chao-Lin Yang b, 1, Jen-Chieh Tsai c, Hao-Yuan Cheng d, **, Wen-Huang Peng a, *
a
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Chinese Medicines, China Medical University, Taichung, 40402, Taiwan
b
Ph.D. Program for Biotechnology Industry, College of Biopharmaceutical and Food Sciences, China Medical University, Taichung, 40402, Taiwan
c
Department of Medicinal Botanical and Health Applications, Da-Yeh University, Dacun, Changhua, 51500, Taiwan
d
Department of Nursing, Chung-Jen Junior College of Nursing, Health Sciences and Management, Chia-Yi City, 62241, Taiwan

A R T I C L E I N F O A B S T R A C T

Keywords: Ethnopharmacological relevance: Uncaria rhynchophylla ([Mi] Jack) (gouteng) exerts antidepressive effects.
Antidepression Rhynchophylline (RH), a major component of U. rhynchophylla, exerts similar pharmacological effects to those of
Rhynchophylline gouteng. Thus, RH may have antidepressive effects.
5-HT
Aim of the study: To investigate the anti-depressive effects of RH in chronic unpredictable mild stress (CUMS)-
BDNF
CREB
induced depressive mice. The anti-depressive mechanism of RH determined by measuring the 5-HT levels, the
expressions of cAMP-response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in
cortex and hippocampus.
Materials and methods: The behaviors of CUMS-induced depressive mice were measured using an open field test
(OFT), forced swimming test (FST), and tail suspension test (TST). 5-HT levels were measured using an ELISA
kits. The expressions of BDNF and CREB were determined using western blot test.
Results: RH increased the frequency of rearing and grooming in the OFT and decreased the immobility time in the
FST and TST. RH effectively increased the 5-HT level and BDNF and CREB expressions in the cortex and
hippocampus.
Conclusion: Our findings indicate that the antidepressive mechanism of RH is related to increased levels of 5-HT
from regulating CREB and BDNF expressions in cortex and hippocampus.

1. Introduction et al., 2019). The action of mechanisms of anti-depressants are mediated

by blocking these changes (Schmidt et al., 2007). Selective serotonin
Depression is a major psychiatric disease in the 21st century (Rehm reuptake inhibitors (Fluoxetine, citalopram) are the commonly used
and Shield, 2019). Depression is caused by a combination of factors, anti-depressants (Zhang et al., 2017). However, anti-depressants cause
including heredity, psychology, and environment. The symptoms of side effects, such as agitation, tremors, anxiety diarrhea, constipation,
depression affect a patient’s family and friends, economic status, daily insomnia, and headaches (Khawam et al., 2006). Herbal medicines
life, physical function, and emotional well-being (Han et al., 2018). should be an alternative treatments to prevent depression.
Changes in the dendritic length and spine density in the cortex and Gouteng (Uncaria rhynchophylla) arrests convulsions, ameliorates
hippocampus are contributed to the neurobiology of depression (Wohleb symptoms of the liver, calms the liver wind, and disperses stagnated
et al., 2016). Depression causes a depletion of monoamines in the brain, liver qi to relieve qi stagnation (Compendium of Materia Medica) which
including serotonin (5-HT), norepinephrine, and dopamine (Hellweg was used in anti-depression (Dai et al., 2022). Studies have demon­
et al., 2007). The treatments for depression are antidepressants (Cuijpers strated that gouteng has a neuroprotective effect which is used to treat

Abbreviations: CUMS, chronic unpredictable mild stress; Rhynchophylline, RH; 5-HT, 5-hydroxytryptamine; CREB, cAMP-response element binding protein;
BDNF, brain derived neurotrophic factor.
* Corresponding author.
** Corresponding author.
E-mail addresses: eland0628@gmail.com (E.-Y. Liu), u105307001@cmu.edu.tw (C.-L. Yang), jenchieh@mail.dyu.edu.tw (J.-C. Tsai), m098026@cjc.edu.tw
(H.-Y. Cheng), whpeng@mail.cmu.edu.tw (W.-H. Peng).
1
All authors contributed equally to this work.

https://doi.org/10.1016/j.jep.2023.116302
Received 6 January 2023; Received in revised form 16 February 2023; Accepted 17 February 2023
Available online 25 February 2023
0378-8741/© 2023 Elsevier B.V. All rights reserved.
E.-Y. Liu et al. Journal of Ethnopharmacology 309 (2023) 116302

epilepsy (Hsieh et al., 2009; Tang et al., 2010), Parkinson’s disease induction and were randomly exposed to one of the following CUMS
(Fujiwara et al., 2006; Shim et al., 2009), Alzheimer’s disease (Hsieh every day of the week: cage tilt (18 h, 45◦ ), fasting (24 h), no water (24
et al., 2010; Jung et al., 2006), anxiety (Kang et al., 2004), and h), day-and-night reversal (24 h), cage crowding, and wet litter. The
inflammation (Yuan et al., 2009). The active pharmacological compo­ stressors were randomly assigned two or three times over the experi­
nents of gouteng is rhynchophylline (RH). The pharmacological effects mental period and varied after the first week of the CUMS test for a total
of RH are similar to those of gouteng (Zhou and Zhou, 2010). RH exerts a of 28 days test (Qu et al., 2020). The nonstressed control mice were
protective effect on inhibiting N-methyl-D-aspartate and 5-HT2 recep­ normally housed in cage (10 mice per cage) in another room. At least 12
tor–mediated neurotoxicity during ischemia. It also affects the levels of h of rest was provided between a stressor and a test to mitigate the ef­
5-HT in the cortex, striatum, hippocampus, and hypothalamus (Hsu fects of acute stress (Pucilowski et al., 1993).
et al., 2012). The anti-depressive-like effect of RH is worthy of study.
BDNF and CREB are associated with numerous psychiatric disorders, 2.4. Experimental protocol
including treatment of mood disorders such as depression (Rasmusson
et al., 2002). BDNF regulation is affected by increasing neuronal acti­ The mice were randomly divided into six groups: the control group,
vation followed by CREB activation. Therefore, the two factors are CUMS group, CUMS + RH 1 mg/kg group, CUMS + RH 5 mg/kg group,
considered to regulate neuronal components (Siuciak et al., 1997). CUMS + RH 25 mg/kg group, and CUMS + fluoxetine 10 mg/kg group.
Whether BDNF and CREB signaling plays a role in the anti-depressive With the exception of the control group, all groups were administered 28
effect of RH needs to be investigated. days of CUMS. RH was dissolved in a 0.5% carboxymethylcellulose so­
CUMS has been widely used to induce depression in animals. It is dium salt solution. In the positive control group, fluoxetine was dis­
considered an appropriate model of the causes of human depression and solved in a 0.9% saline solution. The study design is detailed in Fig. 1.
is widely used in depressive research (Maccari et al., 2001; Mo et al.,
2014; Yu et al., 2014). The depressive symptoms of CUMS mice include 2.5. OFT
loss of pleasure and lack of interest in activities (Willner et al., 1992).
During experiments, stress is induced in mice by manipulating the The mice were performed OFT after being subjected to CUMS
environment, such as by tilting the cage, crowding, water deprivation, (Zueger et al., 2005). A video camera was placed above the box to record
fasting, and day–night reversal (Willner et al., 1987). The OFT is used to mouse rearing and grooming behavior for 6 min. At the end of each
assess mice’s rearing and grooming because CUMS mice exhibit reduced mouse behavioral experiment, the observation box was sprayed with a
frequencies of the two behaviors (Gould et al., 2009). The FST (Porsolt 75% alcohol solution and wiped before the next set of animal behavior
et al., 1977) and TST (Steru et al., 1985) are used to observe mice’s experiments (Gould et al., 2009). All assays were conducted in ten
immobility time. replicates for each experiment.
Therefore, CUMS model was used to examine whether RH would
exert anti-depressive effect by analyzing mouse behaviors in an OFT, 2.6. FST
FST, and TST. The level of 5-HT and the expressions of CREB and BDNF
detected in the cortex and hippocampus were aimed to investigate the Each mouse was performed FST for 6 min according to the method
anti-depressive mechanism of RH. described by Porsolt et al. (1977). During the first 2 min, the mice went
through an adaptation period, and the next 4 min were the immobility
2. Materials and methods time. Two observers recorded the immobility time in which the mice
remained submerged without struggle or only moved to maintain their
2.1. Materials heads above water.

RH (CAS No.76-66-4, 98%) was purchased from ChemFaces (Wuhan, 2.7. TST
China). Beta actin monoclonal antibody was purchased from Proteintech
(Rosemont, IL, USA). Fluoxetine and carboxymethylcellulose sodium Each mouse was performed TST for 6 min according to the method
salt were purchased from Sigma-Aldrich (New York, USA). ST/5-HT described by Steru et al. (1985). The rear end of the mouse tail was taped
(Serotonin/5-Hydroxytryptamine) ELISA Kits were purchased from to an acrylic box 50 cm above the ground. A total of 6 min of experi­
FineTest (Wuhan, China). Anti-CREB and anti-BDNF antibodies were mentation were recorded; the first 2 min constituted adaptation, and the
purchased from Abcam (Cambridge, UK). Phospho-CREB was purchased subsequent 4 min were used to record the immobility time of the mice.
from Cell Signaling Technology (Danvers, MA, USA). Goat antimouse
IgG antibody and goat antirabbit IgG antibodies were obtained from 2.8. Measurement of 5-HT in cortex and hippocampus
Arigo (Hsinchu, Taiwan).
The mice were immediately sacrificed after the TST. The mice’s
2.2. Animals and experimental conditions brains were divided into cortex and hippocampus and weighed. After
weighing, cortex and hippocampus were cut 17.5 mg and 158 μL PBS
Male C57BL/6 mice (BioLASCO, Taipei, Taiwan) weighing 22–24 g solution added for homogenization. After centrifugation for 10 min at
were maintained in the animal center of China Medical University 12,000 rpm and 4 ◦ C, the supernatant was stored in a freezer at − 80 ◦ C.
(Taichung, Taiwan). The mice were kept in 12-h cycles of light and The 5-HT levels of the supernatants of cortex or hippocampus and
darkness. They were provided food and water ad libitum. Ten mice were standard solution were placed in a ST/5-HT ELISA Kit 96 well plate to
maintained in each cage. The Institutional Animal Care and Use Com­ detect the serotonin concentration. The 5-HT levels were recorded and
mittee of China Medical University approved the project (CMU108-S- analyzed using the Thermo Scientific Multiskan Spectrum (Li et al.,
39). All animal experiments were conducted in accordance with the US 2019).
National Institutes of Health’s Guide for the Care and Use of Laboratory
Animals (National Institutes of Health Publication No. 85–23, revised 2.9. Western blot analysis
1996).
The cortex and hippocampus samples were cut 17.5 mg and 158 μL
2.3. Chronic unpredictable mild stress procedure lysis buffer (RIPA, protease inhibitor, and phosphatase inhibitor) was
added for homogenization. After centrifugation for 10 min at 12,000
C57BL/6 mice had a 1-week adaptation period before the CUMS rpm and 4 ◦ C, the supernatant was stored in a freezer at − 80 ◦ C.

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E.-Y. Liu et al. Journal of Ethnopharmacology 309 (2023) 116302

Fig. 1. Experimental design for assessing the effect of RH on depression-like behaviors in CUMS mice. Mice were subjected to CUMS for 28 days. Rhynchophylline
was i. p. injected before 30 min of CUMS.

Western blotting was performed according to the method described than did the untreated CUMS mice (p < 0.05). The CUMS group
by Tzeng et al. (2022) with modification. The supernatants of cortex or exhibited significantly less grooming time in the OFT than did the
hippocampus. The protein expression of CREB, p-CREB, BDNF, and Beta control group (Fig. 2B). The RH 5 mg/kg, RH 25 mg/kg, and fluoxetine
actin in the supernatants of cortex or hippocampus were assessed and groups exhibited significantly increased grooming times in the OFT than
quantified using Gelpro4 software. Beta actin was used as an internal did the CUMS mice (p < 0.05).
loading control (Lu et al., 2020).

2.10. Statistical analysis 3.2. RH on the FST in the CUMS mice

All data are expressed as means ± standard error of the mean (SEM). The CUMS group exhibited significantly longer immobility times
Data were analyzed using one-way ANOVA followed by Scheffe’s mul­ than did the control group during the FST, whereas the RH 5 mg/kg, RH
tiple tests. The criterion for statistical significance was p < 0.05. All 25 mg/kg, and fluoxetine groups exhibited significantly shorter immo­
statistical analyses were performed using SPSS for Windows (IBM, bility times in the FST than did the untreated CUMS group (p < 0.05).
Armonk, NY, USA). The results are presented in Fig. 3.

3. Results
3.3. RH on the TST in the CUMS mice
3.1. RH on the OFT in the CUMS mice
The TST immobility time was significantly longer in the CUMS group
The CUMS groups exhibited significantly lower rearing times in the than in the control group (Fig. 4). The RH 5 mg/kg, RH 25 mg/kg, and
OFT than did the control group (Fig. 2A). The RH 5 mg/kg, RH 25 mg/ fluoxetine groups exhibited significantly shorter immobility times in the
kg, and fluoxetine groups exhibited significantly more rearing behavior TST than did the untreated CUMS group (p < 0.05).

Fig. 2. Effect of RH and fluoxetine intraperitoneal injection on rearing (A) and grooming (B) in OFT. The values are expressed as means ± SEM for each group (n =
10); #p < 0.05 and ###p < 0.001 compared with the control group. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated CUMS group.

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E.-Y. Liu et al. Journal of Ethnopharmacology 309 (2023) 116302

cortex and hippocampus compared with the control group (Fig. 6B and
C). The RH 5 mg/kg, RH 25 mg/kg, and fluoxetine groups exhibited
significantly increased BDNF expressions in the cortex and hippocam­
pus. The expressions of p-CREB in the cortex and hippocampus were
significantly decreased in the untreated CUMS group (Fig. 7B and C).
However, the RH 5 mg/kg, RH 25 mg/kg, and fluoxetine groups
exhibited significantly increased expressions of p-CREB in the cortex and
hippocampus.

4. Discussion

Our previous study demonstrated that an ethanol extract of gouteng

Uncaria rhynchophylla possesses anti-depressive effect (Hsu et al., 2012).
RH is the major component of U. rhynchophylla (Hsu et al., 2012). The
aims of this were intended to investigate the anti-depressive-like effect
of RH and its action of mechanism in CUMS mice. OFTs, FSTs, and TSTs
are commonly used for assessing the anti-depressive-like effect of
various drugs (Bai et al., 2016). In OFTs, the rearing and grooming times
of depressive mice decrease as compared with those of healthy mice
Fig. 3. Effect of RH and fluoxetine intraperitoneal injection on immobility time (Sahin et al., 2019). Our results indicate that the rearing and grooming
in FST. The values are expressed as means ± SEM for each group (n = 10); times of CUMS mice decreased. RH and fluoxetine increased the rearing
###p < 0.001 compared with the control group. **p < 0.01 and ***p < 0.001 and grooming times of CUMS mice. FSTs and TSTs are commonly used to
compared with the untreated CUMS group. study depressive-like behaviors in mice (Perona et al., 2008). In these
two tests, the immobility times of mice are observed. The longer the
mice’s immobility time is, the more severe the mouse’s depressive
symptoms are (Powell et al., 2012). In FST, RH and fluoxetine decreased
immobility time as compared with that of CUMS mice. The same result
was observed in the TST, indicating that RH exerted an
anti-depressive-like effect.
The cortex regulates functions such as feelings of helplessness,
hopelessness, guilt, and suicidal intent in patients with depression (Stahl
and Felker, 2008). Depressed patients exhibit helplessness and a lack of
interest in activity (Wang et al., 2017a, 2017b); this is primarily caused
by decreased activity of the cortex serotonin neurons, resulting in a
decrease in 5-HT concentrations (Abelaira et al., 2013). 5-HT regulates
physiological behavior and many responses such as sleep, appetite, and
hormone secretion (Yohn et al., 2017). 5-HT is closely associated with
neurotrophins, such as BDNF and CREB (Blendy, 2006; Mattson et al.,
2004). BDNF has been demonstrated to play a critical role in neuronal
survival, axonal growth, and synaptic plasticity (Ru et al., 2019). Studies
have reported that a reduction in BDNF may contribute to the depression
caused by CUMS (Liu et al., 2015). The CREB is key in the regulation of
neuroplasticity and neurogenesis (Kandezi et al., 2020). These two
transcription factors play a major role in the cAMP signaling pathway
Fig. 4. Effect of RH and fluoxetine intraperitoneal injection on immobility time that regulates anti-depressive response, neurotransmission, and synaptic
in TST. The values are expressed as means ± SEM for each group (n = 10); plasticity (Hacimusalar and Esel, 2018), both of which aid in neuro­
###p < 0.001 compared with the control group.; *p < 0.05, **p < 0.01, and
plasticity in the brain and thereby increase 5-HT level (Mattson et al.,
***p < 0.001 compared with the untreated CUMS group.
2004). The expressions of BDNF, CREB, and 5HT level are reduced in
depressive mice. 5-HT levels are correlated with BDNF and CREB acti­
3.4. RH on the levels of 5-HT in cortex and hippocampus in the CUMS vation (Wang et al., 2022). Increased BDNF activation potentially in­
mice hibits the pathogenesis of depression (Khan et al., 2019). The CUMS
mice exhibited less rearing and grooming behavior, and exhibited in­
The CUMS group exhibited significantly decreased 5-HT levels in the crease in immobility time in the FST and TST. These results indicate that
cortex than did the control group, whereas the RH 5 mg/kg, RH 25 mg/ the CUMS mice exhibited helplessness and a lack of interest and that the
kg, and fluoxetine groups exhibited significant increases in 5-HT levels 5-HT levels in the cortex of CUMS mice were reduced. The immobility
in the cortex (p < 0.05; Fig. 5A). The untreated CUMS group exhibited a times of the CUMS mice in the TST and FST were associated with
significant decrease in 5-HT in the hippocampus compared with the decrease in level of 5-HT and expression of BDNF and CREB in the
control group, whereas the RH 5 mg/kg, RH 25 mg/kg, and fluoxetine cortex. BDNF and CREB can improve neuroplasticity and increase levels
groups exhibited significant increases in 5-HT levels in the hippocampus of 5-HT to decrease the immobility time of TST and FST in mice with
(Fig. 5B). CUMS-induced depression (Lin et al., 2014). The administration of RH
effectively increased the BDNF and CREB expressions in the cortex of
3.5. Western blot analysis CUMS mice. These findings indicated that RH improved CUMS
mice-induced rearing and grooming behavior, which was associated
The expressions of BDNF and p-CREB were measured using western with reductions in the immobility times in the FST and TST, increased
blot analysis. The results are presented in Figs. 6A and 7A. The untreated levels of 5-HT in the cortex, and enhanced expressions of BDNF and
CUMS group exhibited significantly decreased BDNF expressions in the CREB in CUMS mice.

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E.-Y. Liu et al. Journal of Ethnopharmacology 309 (2023) 116302

Fig. 5. Effect of RH and fluoxetine intraperitoneal injection on the levels of 5-HT in the mice’s cortex (A) and hippocampus (B). The values are expressed as means ±
SEM for each group (n = 10); #p < 0.05 and ###p < 0.001 compared with the control group.; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated
CUMS group.

Fig. 6. Effect of RH and fluoxetine intraperitoneal injection on the levels of BDNF in the mice’s cortex and hippocampus. The graph presents the quantification of the
expression of CREB (A) in the cortex and hippocampus; BDNF in cortex (B); BDNF in hippocampus (C). The values are expressed as means ± SEM for each group (n =
10); ##p < 0.01 and ###p < 0.001 compared with the control group.; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated CUMS group.

Fig. 7. Effect of RH and fluoxetine intraperitoneal injection on the levels of CREB in mice’s cortex and hippocampus. The graph presents the quantification of the
expression of CREB (A) in the cortex and hippocampus; CREB in cortex (B) CREB in hippocampus (C). The values are expressed as means ± SEM for each group (n =
10); ##p < 0.01 and ###p < 0.001 compared with the control group.; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the untreated CUMS group.

The hippocampus regulates memory and neuroplasticity (Sahay and hippocampal synapses caused by the induction of Alzheimer’s disease in
Hen, 2007; Sasmita et al., 2018), Depression can damage the synapses of mice. 5-HT can improve memory (Fu et al., 2021; Buhot et al., 2000). In
the hippocampus, leading to memory impairment (Vythilingam et al., depression models with olfactory bulbectomized rats, antidepressant
2004). Studies have demonstrated that RH repairs the damaged increases BDNF expression in the hippocampus (Liu et al., 2014a, 2014b;

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E.-Y. Liu et al. Journal of Ethnopharmacology 309 (2023) 116302

Wang et al., 2016). The mice lacking CREB exhibit impaired cognitive Fu, W.Y., Hung, K.W., Lau, S.F., Butt, B., Yuen, V.W., Fu, G., Chan, I.C., Ip, F.C.F., Fu, A.
K.Y., Ip, N.Y., 2021. Rhynchophylline administration ameliorates amyloid-β
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