D)STRESS

Formulation Research

This study aimed to assess the sedative-hypnotic effect of the active components of Ziziphus jujuba var. spinosa seed and Polygala tenuifolia root, the possible mechanisms of such effect, and related metabolic pathways. Compared with the control group, high dose Ziziphus jujuba var. spinosa seed and Polygala tenuifolia root (EI30) group and the Clonazepam group were with significantly higher proportions of sleep within 30 min (P = 0.027 and 0.005 respectively).

Compared with the control group, all of the high, medium and low dose of EI30 groups were with significantly shorter sleep latency (P < 0.01) and prolonged sleeping time (P < 0.01). The herbal pair has good sedative-hypnotic effects, although it is weaker than the effect of Clonazepam.

The sedative-hypnotic effect of EI30 is possibly related to the adjustment of neurotransmitters 5-hydroxytryptamine (5-HT), norepinephrine (NE) and dopamine (DA) in the total protein of mice brain tissue. There are five metabolic pathways in vivo most related to the sedative-hypnotic effect of EI30, including the biosynthesis of valine, leucine and isoleucine; metabolism of glyceride; metabolism of alanine, aspartic acid and glutamic acid; metabolism of phenylalanine; and metabolism of cysteine and methionine.

This study reveals the mechanisms of the sedative and hypnotic effects of the herbal pair Ziziphus jujuba var. spinosa seed and Polygala tenuifolia root, by using metabolomics methods. This study provides a basis for further development and utilization of this herbal pair [1].

Ziziphus jujuba var. spinosa seed

Ziziphus jujuba var. spinosa seed, mainly contains substances such as flavonoids, saponins, alkaloids, volatile oils and amino acids [2,3]. Ziziphus jujuba var. spinosa has been proven by previous studies to have the following actions: sedative-hypnotic effect, anti-anxiety effect, anti-depression effect, hypoglycaemic action, anti-dementia effect and the effect of strengthening the immune system. The total flavonoids and total saponins in Ziziphus jujuba var. spinosa are the principal effective components for sedative and hypnotic effects [4].

Sedative and hypnotic effects

One of the main effective compounds for the sedative and hypnotic effects of Ziziphus jujuba var. spinosa seed is spinosin, which is a flavonoid glycoside monomer [5]. Spinosin can induce sleep in mice, prolong the length of pentobarbital induced sleep and reduce sleep latency in rats [6,7]. A previous animal experiment has shown that spinosin has anxiolytic-like effects in mice. High doses of spinosin on mice could significantly increase the number of times entering into the open arms of the elevated plus-maze, the proportion of time spent on the open arms, the number of transitions between light and dark boxes and time spent in the light compartment [8].

Jujuboside A found in Ziziphus jujuba var. spinosa seed had been proven by a previous animal experiment to have sedative effects. A study showed that jujuboside A could reduce the activity of drosophilae melanogaster during the day and prolong their sleep time [9].

Regulation of GABAA and 5-HT Receptors

In this study, a system biology method assisted by UPLC-Q-TOF/MS and RT-qPCR was developed to systematically demonstrate the anxiolytic mechanisms of Ziziphus jujuba var. spinosa seed (SZJ). A total of 35 phytochemicals were identified from SZJ extract (Ziziphus jujuba Mill. var. spinosa [Bunge] Hu ex H.F. Chow), which interact with 71 anxiolytic targets. Protein-protein interaction, genes cluster, Gene Ontology, and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways analysis were subsequently conducted, and results demonstrated that regulation of serotonergic and GABAergic synapse pathways were dominantly involved in the anxiolytic mechanisms of SZJ extract. The effects of SZJ extract on mRNA expressions of multiple GABAA (gamma-aminobutyric acid type A) and 5-HT (serotonin) receptors subtypes were further validated in human neuroblastoma SH-SY5Y cells using RT-qPCR. Results showed that SZJ extract (250 mg/mL) significantly up-regulated the mRNA level of GABRA1 and GABRA3 as well as HTR1A, HTR2A, and HTR2B in non-H2O2 treated SH-SY5Y cells. However, it exerted an inhibitive effect on the overexpressed mRNA of GABRA1, GABRA2, HTR1A, and HTR2A in H2O2 treated SH-SY5Y cells. Taken together, our findings suggest that anxiolytic mechanisms of SZJ mostly involve the regulation of GABAergic and serotonergic synapse pathways, especially a two-way modulation of GABRA1, HTR1A, and HTR2A [10].

Circadian Rhythm and the Serotonergic System

In this study, the hypnotic effect of jujubosides, of Ziziphus jujuba var. spinosa seed, in both day and night period was looked at. It was found that during daytime (9:00-15:00), jujubosides significantly increased the total sleep and rapid eye movement (REM) sleep without significant influence on non-REM (NREM) sleep. During night time (21:00-3:00), jujubosides significantly increased the total sleep and NREM sleep especially the light sleep while showed no significant effect on REM sleep and slow wave sleep (SWS). In pentobarbital-treated mice, jujubosides significantly augmented the hypnotic effect of pentobarbital, proved by increasing sleep time and this augmentative effect was potentiated by 5-hydroxytryptophan. Furthermore, jujubosides inhibited the para-chlorophenylalanine-induced suppression of pentobarbital-induced hypnosis.

These results suggested that the hypnotic effect of jujubosides on normal rats may be influenced by circadian rhythm and the serotonergic system may involve in the hypnotic effect of jujubosides [11].

Ziziphus spinosa bioactive constituents and mechanism of actions [12]

Compound class

Compound name

Potential mechanisms of action

Cyclopeptide alkaloids

Saponin glycosides

Sanjoinines & magnoflorine Jujubosides

GABA-ergic receptor agonists [13]

Modulation of GABAA receptor subunits gene expression [14]

Regulation of the serotonergic system [15, 16, 17]

Inhibition of glutamate-mediated pathways [18]

Jujuboside metabolite

Flavones

Jujubogenin (JuAm)

Spinosin

Modulation of GABAA receptors [19,20,21]

Modulation of GABAA receptors [22]

Serotonergic mechanism and antagonism of 5-HT1A receptors [23,24]

Abbreviations: 5-HT1A: 5-hydroxytryptamine(1A); GABA: gamma-aminobutyric acid.

Main pharmacological actions for Ziziphus jujuba seeds [25]

Pharmacological

actions

Active pharmacological compounds

Mechanism involved

Anxiolytic and hypnotic-sedative

Saponins such as Jujuboside A (JuA) and jujuboside B (JuB) or probably jujubogenin. Sanjoinine A

Flavonoids (Spinosin and others).

GABAA, GABA-binding conformation, modulation.

GABA-benzodiazepine receptor activation, increased GABA synthesis via GAD65/67 activation and influence over GABA receptor subunits composition.

Postsynaptic 5-HT1A receptor-dependent mechanism

Neural damage protection

JuA

Binding action with calmodulin, interrupting the signal process intrigued by NMDA calcium efflux or an inhibitory excitatory effect involving presynaptic mechanism.

Anti-inflammatory

Phenolic compounds, oxygenated mono- and sesquiterpenes, and their respective hydrocarbons. Epigallocatechin, gallocatechin, spinosin, 6”’feruloylspinosin and 6”’sinapoylspinosin.

An action mediating calmoduline

Inhibition of NF-κβ DNA binding and decreased nuclear transcription of NF-κβ-p65.

Dyslipidaemia

C-28 carboxilic acid triterpenoids such as oleanonic acid, pomolic acid, and pomonic acid

Total cholesterol, atherogenic index, LDL, free cholesterol, triglyceride, phospholipid and blood glucose in serum reduction. Inhibition of foam cell formation.

Learning and memory performance

Spinosin, JuA, JuB and others

The possible mechanisms of JuA seem to be largely related to its antioxidant activity and the de- creasing activation of caspase-9 and caspase-3, resulting in a reduction of cell apoptosis.

Hair growth promoting

Fatty acids

The exact mechanism of the stimulation of hair growth is not known, but fatty acids might be involved.

 Polygala tenuifolia root

Polygala tenuifolia root mainly contains substances such as saponins, sugar esters, volatile oils, organic acids, and small amounts of alkaloids and flavonol glycosides. Polygala tenuifolia root is reported to have effects of sedation and hypnosis, memory-enhancing, anti-dementia, anti-aging, and anti-inflammatory [26].

Sedative and hypnotic effects

Senegenin is the active component for the sedative and hypnotic effects of Polygala tenuifolia root [27]. Tenuifolin, a kind of senegenin, has been proven to have sedative effects. In a study, mice were intragastrically injected with tenuifolin at doses of 20, 40, and 80 mg/kg, and the results showed that 40 mg/kg and 80 mg/kg doses groups were with significantly increased the non-rem sleep time, rem sleep time, and total sleep time [28].

Other substances in Polygala tenuifolia root had been proven to have sedative effects as well. The sedative-hypnotic effects of 3,4,5-Trimethoxycinnamic acid (TMAC), a compound derived from Polygala tenuifolia root, were proven to act via increasing the chloridion influx and activating glutamic acid decarboxylase and γ-subunit of GABAA receptors in the cerebellar granule cells [29].

Insomnia

The beneficial effects for sleep of the herb Polygala tenuifolia and Alprazolam were investigated, and the results indicated that both could benefit sleep via the shortening of sleep induction time and the increase in wave forms of electroencephalogram (EEG) associated with stages 1, 2 and 3 of the non-rapid eye movement (NREM) sleep. Increase of numbers of theta waves, v waves, delta waves and k complex were observed in mice, indicating deeper and better quality of sleep. There was an obvious increase in the duration of theta plus delta waves in both Polygala and Alprazolam treated groups, while the wave duration was slightly longer in the Polygala group than the Alprazolam group. When the Alprazolam treated group was added with Polygala, the theta plus delta wave duration increased and exceeded that from Alprazolam treatment alone. Similarly, increase of duration of k complex was also observed in both Polygala and Alprazolam treated groups. This study illustrated that the herbal agent Polygala tenuifolia could have at least similar efficacy in the induction of sleep as the manufactured drug Alprazolam [30].

Depression and Chronic stress

This study aimed to explore the anti-depressant effects of Senegenin (SEN) on behavioural changes and inflammatory responses in mice induced by chronic un-predictable mild stress (CUMS). SEN treatment remarkably ameliorated CUMS-induced behavioural abnormalities, such as improving locomotor activity, decreasing immobility time in Tail suspension test (TST) and Forced swimming test (FST), and increasing sucrose intake in Sucrose preference test (SPT). Additionally, SEN improved protein levels of Brain-derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3) expression. In response to stress, p65 was activated to promote production of pro-IL-1β, and then cleaved to mature IL-1β by NOD-like receptor protein 3 (NLRP3) inflammasome pathway in hippocampus of CUMS mice. After SEN treatment, protein activation related to NLRP3 inflammasome pathway was down-regulated, which inhibited IL- 1β secretion. These results demonstrate that SEN plays an important role in treatment CUMS-induced depression in mice, possibly via suppression of pathway activation associated with NLRP3 inflammasome [31].

Note: Several studies have found that stress levels influence the association between sleep and BDNF levels, and sleep quality interferes directly in the relation of stress with BDNF levels. Insomniacs present decreased serum BDNF levels and sleep problems are related to impaired BDNF synthesis [32, 33].Neurotrophin-3 (NT-3) is a neurotrophic factor closely related to nerve growth factor which is capable of modulating neuronal activity. NT-3 rapidly modulates the activity of NPO neurons involved in REM sleep. Cholinergic neurons in the LDT and PPT contain NT-3. Taken together it is plausible that NT-3 is involved in the control of naturally occurring REM sleep [34].

Rapid-onset anti-depressant activity

Polygala tenuifolia can protect against N-methyl D-aspartate (NMDA) neurotoxicity and induce brain-derived neurotrophic factor (BDNF) expression, suggesting modulatory roles at glutamatergic synapses and possible antidepressant action.

In this preclinical rodent study, Polygala tenuifolia demonstrated antidepressant-like effects in 8-week-old male C57Bl/6 mice by decreasing behavioural despair in the forced swim and tail suspension tasks and increasing hedonic-like behaviour in the female urine sniffing test 30 minutes after a single oral administration of 0.1 mg/kg. Reduced latency to acquire a food pellet in the novely suppressed feeding paradigm, without change in anxiety-like behaviours suggested a rapid-onset nature of the antidepressant-like effect. Therefore, Polygala tenuifolia exerted rapid-onset antidepressant effects by modulating glutamatergic synapses in critical brain circuits of depression.

Immobility reduction in tail suspension task was blocked by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist NBQX, a pattern previously demonstrated by ketamine and other ketamine-like rapid-onset antidepressants. Also similarly to ketamine, Polygala tenuifolia appeared to acutely decrease phosphorylation of GluR1 serine-845 in the hippocampus while leaving the phosphorylation of hippocampal mTOR serine 2448 unchanged.

Polygala may possibly substitute for other rapid-onset antidepressants like ketamine that are associated with addiction risk and unacceptable side effects [35].

Depression and HPA axis

3,6’-Disinapoyl sucrose (DISS) is an active oligosaccharide ester component from roots of Polygala tenuifolia, and its antidepressant effects was found in the forced swimming test (FST) and tail suspension test (TST). The aim of this study was to review the antidepressant effects of DISS in the chronic unpredictable mild stress (CMS) model and explore the underlying mechanisms in the hypothalamic-pituitary-adrenal (HPA) axis. They found that when subjected to the chronic stress protocol for 28 days, animals showed reduced sensitivity to reward and abnormality in the HPA axis. DISS improved the reward reaction as measured by increasing sucrose consumption, remarkably reduced serum CORT, ACTH and CRH levels in the CMS-treated rats. In addition, DISS enhanced the expression of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNA. These results indicated that the antidepressant effects of DISS in chronically stressed animals might relate to the modulating effects on the HPA axis, which might be an important mechanism for its antidepressant effect [36].

Anxiety and HPA axis

The present study investigated the effects of the administration of Polygala tenuifolia (PT) on repeated restraint stress–induced anxiety-like behavioural responses in mice. Additionally, possible changes in the central noradrenergic and brain-derived neurotrophic factor (BDNF)–signalling systems were assessed in the hippocampus of these mice. Compared with control subjects, the daily administration of higher doses of PT increased open-arm exploration in an elevated plus maze and the total number of line crossings in an open-field test. PT also reversed the increased expression of tyrosine hydroxylase in the locus coeruleus and the decreased expression of BDNF mRNA in the hippocampus. Together, these findings demonstrate that the administration of PT prior to repeated restraint stress significantly reduced anxiety-like behaviours. These changes were associated with a modification of the central noradrenergic system and the upregulation of BDNF expression, which in turn attenuated activity in the HPA axis [37].
Neurotrophic Effects of SenegeninTing et al., (2013) studied the neurotrophic effects of senegenin on the expression of MAP2 mRNA and BDNF mRNA in cultured cerebral cortical neurons. LDH assay showed that senegenin at the concentration of 0. 5 micromol/L,1 micromol/L and 2 micromol/L could obviously enhance the survival of cells and the survival rates were in dose-dependent manner to some extent. Moreover, the low, medium and high concentrations of senegenin significantly increased the expression of MAP2 mRNA and BDNF mRNA.
Their study suggests that suitable dose of senegenin can increase the expression of MAP2 mRNA and BDNF mRNA in cerebral cortical neurones [38].
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