Ethanol extract of the seed of Zizyphus jujuba var. spinosa potentiates hippocampal synaptic transmission through mitogen-activated protein kinase, adenylyl cyclase, and protein kinase A pathways

doi:10.1016/j.jep.2017.02.009

Journal of Ethnopharmacology

Volume 200, 22 March 2017, Pages 16-21

https://doi.org/10.1016/j.jep.2017.02.009 Get rights and content

Abstract

Ethnopharmacological relevance

As the seed of Zizyphus jujuba var. spinosa (Bunge) Hu ex H.F. Chow (Rhamnaceae) has been used to sleep disturbances in traditional Chinese and Korean medicine, many previous studies have focused on its sedative effect.

Aim of the study

Recently, we reported the neuroprotective effect of the effect of Z. jujuba var. spinosa. However, its effects on synaptic function have not yet been studied. In this project, we examined the action of ethanol extract of the seed of Z. jujuba var. spinosa (DHP1401) on synaptic transmission in the hippocampus.

Materials and methods

To investigate the effects of DHP1401, field recordings were conducted using hippocampal slices (400 µm). Object recognition test was introduced to examine whether DHP1401 affect normal recognition memory.

Results

DHP1401 (50 μg/ml) induced a significant increase in synaptic activity in Shaffer collateral pathway in a concentration-dependent manner. This increase of synaptic responses was blocked by NBQX, a broad spectrum α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist, but not IEM-1460, a Ca²⁺-permeable AMPAR blocker. Moreover, U0126, a mitogen-activated protein kinase inhibitor, SQ22536, an adenylyl cyclase inhibitor, and PKI, a protein kinase A inhibitor, blocked DHP1401-induced increase in synaptic transmission. Finally, DHP1401 facilitated object recognition memory.

Conclusions

These results suggest that DHP1401 increase synaptic transmission through increase of synaptic AMPAR transmission via MAPK, AC and PAK.

Graphical abstract

Introduction

The seeds of Zizyphus jujuba var. spinosa (Bunge) Hu ex H.F. Chow (Rhamnaceae) (English name: spine date seed) have been used as a hypnotic agent (Zhu, 1998) in traditional Chinese and Korean medicine. Recent studies revealed that the seed of the Ziziphus species produced sleep state without inducing convulsion or muscle relaxation (Peng et al., 2000). Moreover, as active constituents, jujuboside A and jujubogenin produced a hypnotic effect through modulations in neurotransmitter systems, such as serotonin and GABA systems (Chen et al., 2008, Wang et al., 2012, Wang et al., 2015, You et al., 2010). However, its precise action mechanism on the brain is still not clear.

Glutamate receptors including N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) are known to play a crucial role in learning and memory (Lussier et al., 2015, Sanderson et al., 2008, Tsien et al., 1996). NMDAR activation causes AMPAR translocation into postsynaptic membrane and this causes long-term potentiation, a cellular model of learning and memory (Bliss and Collingridge, 2013, Lu et al., 2001, Sanderson et al., 2008). The GABAergic system can modulate the actions of these glutamate receptors by limiting postsynaptic depolarization (McBain and Fisahn, 2001, Pouille and Scanziani, 2001, Staley and Mody, 1992). Therefore, many drugs showing sedative effects, including diazepam, a typical hypnotic agent, have been reported to impair memory processing (Kim et al., 2012, Kim et al., 2009). In line with this, the seed of Z. jujuba var. spinosa may be speculated to produce amnesic effects. However, we recently found memory-enhancing effects of the seed of Z. jujuba var. spinosa and its constituents, including spinosin and swerticin (Jung et al., 2014, Ko et al., 2015, Lee et al., 2016a, Lee et al., 2013, Lee et al., 2016b). To better understand this discrepancy, in the present study we examined the effect of the seed of Z. jujuba var. spinosa on synaptic transmission in the hippocampus. Interestingly, we found that ethanol extract of the seed of Z. jujuba var. spinosa facilitated basal excitatory transmission and this might have been mediated by mitogen-activated protein kinase, (MAPK) adenylyl cyclase (AC), and protein kinase A (PKA) pathways.

Section snippets

Animals

We purchased male CD-1 mice (26–28 g, 6 weeks old) from the SAMTAKO biokorea (Osansi, Korea). Mice were housed in the Dong-A University Animal Care Unit for 1 week before the start. Four mice were contained in a cage. Mice were allowed freely access to water and food (temperature: 23±1 °C; humidity: 60±10%; light/dark cycle: from 07:30 to 19:30). Institutional Animal Care and Use Committee of Dong-A University (Korea) approved the protocols of animal experiments.

Materials

A standardized ethanol extract of

DHP1401 induces facilitation of synaptic transmission

To test the effect of DHP1401 on synaptic transmission, we applied DHP1401 directly to hippocampal slices and found that DHP1401 induced an immediate and lasting facilitation of fEPSP on the hippocampus in a concentration dependent manner (F_{2, 12}=10.69, P<0.05, Fig. 1A and B.

DHP1401-facilitation of synaptic transmission is AMPA receptor dependent

To isolate the component of DHP1401-facilitation of synaptic transmission, receptor antagonism study was carried out. fEPSP was enhanced by DHP1401 in the presence of NMDA receptor antagonist D-AP5 (136±4.9%, n=5. Fig. 2A),

Discussion

Studies in the last few years have indicated that the seed of Z. jujuba var. spinosa and its constituents has beneficial effects in various dementia models. However, its precise mechanisms of action are still not clear. In the present study, we found that DHP1401, a standardized ethanol extract of the seed of Z. jujuba var. spinosa facilitated basal excitatory synaptic transmission. We also found that NBQX blocked the DHP1401-induced enhancement of the CA1 fEPSP responses, which indicates that

Author contributions

J.Y., J.H.R., and D.H.K. designed all of the experiments together. S.Y.J. and J.H.Y. performed in vitro electrophysiology. I.H.J. and T.J.C. prepared DHP1401 and standardized. S.L. and J.W.J. performed in vivo behavioral experiment. Y.C.L., J.H.R., and D.H.K. wrote manuscript.

Acknowledgement

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2016R1A5A2007009) and by the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI16C0172).

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var. spinose (Bunge) Hu ex H. F. Chou, which has several pharmacological effects such as neuroprotection and cardiovascular protection [12,13]. Studies have shown that ZSS and Chinese herbal formulas containing ZSS are beneficial to the central nervous system and have obvious antidepressant effect [14–17].
The aim of this study was to investigate the antidepressant effect of Jujuboside A (JuA) on corticosterone (CORT)-induced depression in mice and explore the underlying mechanisms.
The mice models were submitted to CORT and treated with JuA (10 and 30 mg/kg) for three weeks. Experiments were also performed on mice with brain-derived neurotrophic factor knockdown (BDNF (±)) as control subjects. Behavioral tests, including the open field test (OFT), tail suspension test (TST), forced swimming test (FST) and Morris water maze (MWM), were then performed to evaluate the antidepressant effect of JuA. The expression levels of BDNF, tyrosine kinase receptor B (TrkB), and cyclic AMP response element binding protein (CREB) in the hippocampi of mice were examined by immunohistochemistry (IHC) and Western blot. The effect of JuA on the viability of mouse hippocampal cells (HT22) was also assessed by CCK-8 assay.
JuA significantly decreased the OFT and TST immobility time of the mice, the total distance travelled and the time spent in the central area also effectively increased in the OFT. In the MWM, the escape latencies of the mice decreased remarkably, while the number of times the mice crossed the platform and the target quadrant increased significantly after treatment with JuA. In addition, the BDNF, TrkB, and CREB expression levels were significantly increased in the hippocampi of the mice treated with JuA. Furthermore, JuA clearly attenuated CORT-induced cell injury, as evidenced by the increased viability of the HT22 cells.
These findings demonstrated that JuA may exhibit potential antidepressant effect in mice by increasing protein expression levels of BDNF, TrkB, CREB, and improving the viability of the hippocampal cells.
Inhibitory effects and molecular mechanism on mushroom tyrosinase by condensed tannins isolation from the fruit of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow
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spinosa (Bunge) Hu ex H. F. Chow) is a deciduous shrub or small tree with a high nutritional and medicinal value, contains a variety of bioactive substances, such as Sour jujube polysaccharide, flavonoids, saponins, triterpenoids, alkaloids, cAMP, CGMP [9–11]. Several substances of jujube kernel, a mature seed of dried jujube plants have been extracted [12–18]. However, there are few reports concerning the sour jujube fruit's bioactivities, in particular from the fruit's tannin.
The structure of extracted condensed tannin (CT) from the fruit of Sour jujube (Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow) and the molecular mechanisms by which CT inhibits the activity of mushroom tyrosinase were investigated. The structure of CT was characterized by high performance liquid chromatography electrospray ionization mass spectrometry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The kinetic assays were used to detect inhibition effect, type and mechanism. UV scanning, fluorescence quenching, copper interacting, o-quinone interaction and molecular docking assays were also used to reveal the molecular mechanisms by which CT inhibit tyrosinase. The results showed the structural units of CT containing afzelechin/epiafzelechin, catechin/epicatechin, and gallocatechin/epigallocatechin. Kinetic analysis showed that CT inhibits both the monophenolase and diphenolase activities of tyrosinase and exhibits reversible, mixed type mechanism. The fruit CT interacts primarily with the copper ions and specific amino acid residue (Asn191, Thr203, Ala202, Ser206, Met201, His194, His54, Glu182 and Ile42) in the active site of tyrosinase to disturb oxidation of substrates by tyrosinase. These results suggested the sour jujube fruit is a potential natural source of tyrosinase inhibitors, and has a potential to be used in food preservation, whitening cosmetics.
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Specifically, the ethanol extract of Zizyphus jujuba var. spinosa seeds has shown improvement in such cholinergic blockade-induced cognitive impairments in mice [6] and amelioration the memory deficits in an Alzheimer’s disease mouse model by elevating the plasmin level [7,8]. A standardized dried 50% ethanolic extract of these seeds that contains spinosin as a marker component is currently under development for the treatment of Alzheimer’s disease in Korea under the name DHP1401 (Daehwa Pharmaceutical Co., Ltd, Sungnam, Republic of Korea).
Spinosin, which is traditionally used for sedation and sleep disorders, has recently shown potential effects in alleviating memory loss. As spinosin is the main bioactive component in a standardized dried 50% ethanol extract of the seeds of Zizyphus jujuba var. spinosa, a Phase IIb clinical trial is ongoing, in Korea for the combination of the above extract formulated in a tablet (DHP1401 tablet) with donepezil hydrochloride (Aricept® tablet) in patients with mild to moderate Alzheimer’s disease. Therefore, to promote safety and efficacy evaluations, a reliable method for the simultaneous detection and analysis of the two drugs is needed. Toward this end, in this study, we established and validated a rapid and sensitive LC–MS/MS method for the simultaneous determination of donepezil, its pharmacologically active metabolite 6-O-desmethyl donepezil, and spinosin in beagle dog plasma (50 μL). After optimization of the system, we used methanol for simple protein precipitation. Chromatographic separation was performed using a Phenomenex Luna C₁₈ column (100 × 2.0 mm, 3 μm) with a mobile phase consisting of 0.1% formic acid in acetonitrile-0.1% formic acid in distilled water (2:8, v/v) at a flow rate of 0.65 mL/min. All analytes were detected and quantified in selected reaction monitoring mode. All calibration curves showed good linearity (r ≥ 0.9965) over the concentration range of 0.02–20, 0.02–10, and 0.5–250 ng/mL for donepezil, for 6-O-desmethyl donepezil, and spinosin, respectively. This validated method was then successfully applied to a pharmacokinetic study in beagle dogs with no evidence for potential drug–drug interactions between DHP1401 and donepezil hydrochloride. This information and optimized assay can be useful for the anticipated co-administration of these two drugs in clinical settings.
Wild Jujube (Ziziphus jujuba var. spinosa): A Review of Its Phytonutrients, Health Benefits, Metabolism, and Applications
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¹: These authors are equally contributed in this study.

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Ethanol extract of the seed of Zizyphus jujuba var. spinosa potentiates hippocampal synaptic transmission through mitogen-activated protein kinase, adenylyl cyclase, and protein kinase A pathways

Abstract

Ethnopharmacological relevance

Aim of the study

Materials and methods

Results

Conclusions

Graphical abstract

Introduction

Section snippets

Animals

Materials

DHP1401 induces facilitation of synaptic transmission

DHP1401-facilitation of synaptic transmission is AMPA receptor dependent

Discussion

Author contributions

Acknowledgement

Pharmacol. Biochem. Behav.

Neuron

Behav. Brain Res.

Pharmacol. Biochem. Behav.

Neuron

J. Biol. Chem.

Prog. Neurobiol.

J. Ethnopharmacol.

Prog. Brain Res.

Neurobiol. Learn Mem.

Cell

J. Proteom.

J. Ethnopharmacol.

Behav. Brain Res.

J. Ethnopharmacol.

Neurobiol. Learn Mem.

Chronic caffeine treatment prevents stress-induced LTP impairment: the critical role of phosphorylated CaMKII and BDNF

J. Mol. Neurosci.

Effects of stress and hippocampal NMDA receptor antagonism on recognition memory in rats

Learn Mem.

Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase

J. Neurosci.

Synaptic modifications in cultured hippocampal neurons: dependence on spike timing, synaptic strength, and postsynaptic cell type

J. Neurosci.

A synaptic model of memory: long-term potentiation in the hippocampus

Nature

Expression of NMDA receptor-dependent LTP in the hippocampus: bridging the divide

Mol. Brain