Anxiety is characterized as excessive and persistent worry about the future, which in turn can impact one’s ability to carry out activities of daily living. Anxiety can be divided into generalized anxiety disorder, panic disorder, obsessive-compulsive disorder, social anxiety disorder, and posttraumatic stress disorder (Möhler, 2012; Cohen et al., 2015). Physiological anxiety symptoms include pounding heart, difficulty breathing, upset stomach, muscle tension, sweating, and feeling faint or shaky (Teychenne et al., 2015). The global prevalence of anxiety is estimated at 16.6% across the life span, and it becomes a burden of healthcare and quality of life (Somers et al., 2006); therefore, it is important to develop effective and safe solutions for treatment (Starcevic, 2006). Tricyclic antidepressants, serotonin-specific reuptake inhibitors, and benzodiazepines have been developed to mitigate anxiety. While effective, these classes of drugs come with many side effects, such as insomnia, sexual dysfunction, suicidal ideation, and/or drug-dependency (Lakhan and Vieira, 2010). Therefore, the use of complementary and alternative medicines to improve anxiety has received increased attention. Traditional medicinal materials, such as Jujube seed (Ziziphus jujuba Mill., Rhamnaceae) (SZJ) (Huang et al., 2008; Wang Y. et al., 2008), saffron (Crocus sativus L.) (Lopresti et al., 2018; Milajerdi et al., 2018), valerian root (Valeriana officinalis L.), and passion flower (Passiflora incarnata L.) (Müller et al., 2003; Cass, 2004; Benke et al., 2009) have shown anti-anxiety benefits.

SZJ, also known as Suanzaoren in Chinese, was first recorded in Shennong Bencao Jing, the earliest classic treatise of Chinese Materia Medica. SZJ has a long history of use in China as a vital food and/or medicine that traditionally is considered to sustain human health by calming the mind and improving the quality of sleep. In recent years, accumulated evidences have shown that SZJ and/or its preparations exert positive outcome on insomnia (Jiang et al., 2007; Chen et al., 2011; Ni et al., 2015; Shergis et al., 2017; Xiao et al., 2018), anxiety (Peng et al., 2000; Liu et al., 2015), and depression (Liu et al., 2012; Liang et al., 2016), mainly through regulating GABAergic (Chen et al., 2007; Chen, 2008; Shergis et al., 2017) and serotoninergic systems (Wang L.-E. et al., 2008; Wang et al., 2010; Liu et al., 2015). Jujubosides (e.g., jujuboside A, B), C-glycoside flavonoid (e.g., spinosin), and pentacyclic triterpenic acid (e.g., betulinic acid) have been identified from SZJ (Liu et al., 2007; Zhang et al., 2008; Lee et al., 2016) as the potential active phytochemicals contributing to these healthy benefits.

Although previous studies have shown promising anxiolytic effects of SZJ, the underlying mechanism has not been systematically and comprehensively investigated. In our current work, we developed an integrated strategy of system biology assisted by ultra-performance liquid chromatography quadrupole-time of flight mass spectrometer (UPLC-Q-TOF/MS) and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) to uncover the active phytochemicals and anxiolytic mechanism of SZJ extract. This approach provides a modern and practical way to study complicated chemical systems with multiple pathways and connected targets, which is otherwise a difficult challenge in mechanistic research of traditional Chinese medicine ingredients.

The identification of phytochemicals in herbal materials is a critical step during the process of system biology analysis. Herbal materials are often subjected to extraction, concentration, and/or purification, resulting in the phytochemical compositions alteration. The phytochemical data from current databases (e.g., TCMSP, http://tcmspw.com/tcmsp.php; TCMID, http://www.megabionet.org/tcmid/) may not be used directly for system biology investigation. Additional methods for phytochemical identification, such as UPLC-Q-TOF/MS, should be a complementary tool to obtain more accurate results of phytochemical compositions (Shen et al., 2013). In current work, a series of flavonoid glycosides and saponins were identified from SZJ extract, in which the spinosin derivatives including 6’’’-vanilloylspinosin, 6’’’-para-hydroxylbenzoylspinosin, 6’’’-sinapoylspinosin, 6’’’-para-coumaroylspinosin, 6’’’-feruloyspinosin, 6’’’-(-)-phaseoylspinosin, and 6’’-O-feruloylspinosin are rare in other plant species. The studies on their bioactivities and effective targets have been so poorly reported that there is not enough data for system biology analysis. In addition, their chemical structures are complicated and contain multi-chiral centers that bring a great challenge to obtain the potential targets through reverse virtual fishing technique. Traditional system biology analysis would take ADME (absorption, distribution, metabolism, and excretion) screening strategy that may exclude these glycosides with low oral bioavailability and low drug-likeness (Yue et al., 2017a; Yue et al., 2017b). For instance, ginsenosides are the dominant phytochemicals in ginseng and are thought to be contributed to its multiple bioactivities (Ru et al., 2015; Kim et al., 2017). However, the above analysis strategy, i.e., ADME screening, would exclude the ginsenosides along with their contributions on efficacy when performing systematic research of ginseng. Therefore, such an analytical strategy is incomplete and not systematic. In fact, it has been well demonstrated that the metabolites of the ginsenosides are responsible for the specific bioactivities (Chen et al., 2018; Kim, 2018). Similarly, the chemoinformatics and pharmacoinformatics approach indicated that jujubogenin was the effective GABA_A agonist, neither jujuboside A nor jujuboside B (Chen, 2009). Gut microbes play an important role in favoring phytochemicals transformation into metabolites endowed with biological activity (Dey, 2019). As a result, the strategy that involves the metabolites of glycosides in gastrointestinal environment (e.g., gut microbes) will be a more reasonable approach to understand the actual efficacy and mechanism of herbal materials in which the glycosides are considered as the main active components.

The GABA_A receptors are chloride channels and are composed of several subunit classes (α, β, γ, δ, and ϵ) (Olsen and Sieghart, 2008). GABAergic neurotransmission plays an important role in anxiety status. Previous studies have shown that deficit of GABA_A receptors and reduction of GABA transmission were observed in people with anxiety-like symptoms (Horowski and Dorow, 2002; Nutt and Malizia, 2004; Hasler et al., 2008). In contrast, positive modulation of GABA_A receptors and enhancement of GABA transmission have shown anxiolytic effects. Classic benzodiazepines reduce anxiety by interacting with the GABA_A receptors via the benzodiazepine binding site, which is present at the interface of α1, α2, α3, or α5 subunits and γ subunit of GABA_A receptors (Möhler, 2012). Other classes of compounds, GABA, barbiturates, and alcohol also could act at different benzodiazepine binding sites to increase the opening of the chloride channel resulting in enhancement of inhibitory synaptic transmission (Harris, 1990; Schousboe and Redburn, 1995). The results of our system biology study suggested the GABA_A receptors signaling is a significant pathway involving in anxiolytic effect of SZJ. In fact, pharmacologic study has found that spinosin, a major C-glycoside flavonoid in SZJ, exerted anxiolytic-like effects via modulation of GABA_A and 5-HT receptors (Liu et al., 2015). Similarly, 6′′′-feruloylspinosin and spinosin have been reported to significantly enhance the expression of GABRA1 and GABRA5 mRNA in rat hippocampal neurons (Qiao et al., 2016). In addition, it has been found that stimulation of jujuboside A at 50 µg/mL could increase the mRNA transcription levels of GABRA1, GABRA5, GABRB1, and GABRB2 in hippocampal neurons (You et al., 2010; Wang et al., 2015); however, long time stimulation of jujuboside A at a high dose of 100 µg/mL result in the decrease of GABRA1 and GABRB2 mRNAs expression (You et al., 2010). These results suggested a two-way modulatory effect of SZJ on GABRA1 and other GABA_A receptors. Such benefits were similar to what we found in this work, that is, SZJ extract enhanced mRNA level of GABRA1 in non-H₂O₂ treated SH-SY5Y cells, but inhibited the H₂O₂-induced overexpression of GABRA1. Therefore, combining with the results from literatures and our results, it was suggested that SZJ exhibited anxiolytic effects through modulating GABA_A receptors, in which a two-way modulation of GABRA1 may play an important role.

It was well established that the alteration of various behaviors in anxiety disorders including appetite, mood, sleep, and cognitive function have been linked to the serotonergic system (Liu Y. et al., 2018; Liu et al., 2019). Serotonin receptors are prevalent throughout the nervous system and the periphery, and they potentially control the serotonergic neurotransmission throughout the brain and neuronal activity to alleviate neuropsychiatric disorders (Okazawa et al., 1999). Generally, the activation of HTR1A, HTR2A receptors can produce anxiolytic effects, whereas inactivation of them increases anxiety-like behaviors (Clinard et al., 2015; Spiacci et al., 2016). Involvement of other 5-HT receptors including HTR1B, HTR1B, and HTR2C in the mechanisms of anxiety have also been recognized (Graeff et al., 1996; Griebel et al., 1997; McCorvy and Roth, 2015). Similarly, our system biology analysis found that the serotonergic synapse pathway was dominant in anxiolytic effects mechanism of SZJ extract, in which different subtypes of 5-HT receptors were involved. Notably, as the same effect on GABRA1, SZJ extract also showed a two-way modulation on HTR1A and HTR2A in our RT-qPCR test. Genetic studies in animal models have suggested that anxiety-like behavior can increase when the HTR1A function is eliminated or overexpressed (Overstreet et al., 2003). Hence, these results suggested the involvement of modulating serotonergic synapse pathway, specifically two-way modulation of HTR1A and HTR2A in anxiolytic effects of SZJ.

In addition, the cannabinoid receptors (CNR) are extensively expressed in areas of the nervous system and have been found closely associated with anxiety behavior (Akirav, 2011). It has been well illustrated that endocannabinoid (eCB) reduces the serotonin release in the central nervous system and increases the expression and function of HTR1A in the hippocampus via the activation of CNR1 (Haj-Dahmane and Shen, 2011; Patel et al., 2017). Beyond CNR1, eCB system could exert actions on other targets including CNR2, transient receptor potential vanilloid receptor type 1 (TRPV1), or cyclooxygenase-2 (COX2) to participate in improvement of anxiety (Patel et al., 2017). In addition, cyclic AMP-responsive element-binding protein (CREB) has been suggested to be crucial for the role of HTR1A in modulating anxiety-related behaviors via mediating hippoacampus structural plasticity (Zhang J. et al., 2016). Intriguingly, this systematic analysis work showed phytochemicals in SZJ extracts potentially act on the above mentioned targets including CNR1, CNR2, TRPV1, COX2, and CREB. These results, to some extent, suggested that the mechanism of action of SZJ in anti-anxiety may also involve those pathways/targets that indirectly modulate eCB and serotoninergic systems. More attention needs to be paid to those targets/pathways in further experimental studies on anxiolytic effects of SZJ.

The phytochemicals in herbal medicines are the substantial basis for their pharmacologic actions. Those phytochemicals with good bioactivity and high content are considered to be the chemical markers in quality control of the herbal medicines. Jujuboside A and spinosin are used to quality markers of SZJ crude drug in Chinese Pharmacopoeia (Edition 2020). Combining the results from literatures reports (Han et al., 2009; Abdoul-Azize, 2016) and our results, the modulations of GABAergic and serotoninergic systems seem the major mechanisms of SZJ exerting anxiolytic effects, as well as the traditional efficacy of nourishing heart and calming mind. Based on that, we abstracted the phytochemicals-targets-pathway sub-networks of GABAergic and serotoninergic synapse pathways. As shown in Figure 7, it demonstrated that metabolites of C-glycosides (spinosin, etc.) and jujubosides (jujuboside A, etc.) including apigenin, kaempferol, naringenin, genkwanin, and jujubogenin were involved in modulation of GABAergic and serotoninergic synapse pathways. The result provides the extra evidence to support that C-glycosides and jujubosides are responsible for the anxiolytic effects of SZJ, and they support jujuboside A and spinosin as chemical markers for quality control of SZJ and its preparations. Beyond the C-glycosides and jujubosides, the involvements of triterpenic acid (e.g., betulinic acid) and alkaloid (zizyphusine) were also observed in modulation of GABAergic and serotoninergic synapse pathways. Specifically, betulinic acid is a function of the modulating GABAergic system via multiple subtypes of GABA_A receptors, whereas zizyphusine is a function of the modulating serotoninergic system via multiple subtypes of 5-HT receptors. Notably, it has been reported that zizyphusine was identified as one of the principal components in SZJ by principal component analysis (Sun et al., 2014). And according to records in TCMIP, zizyphusine is exclusively derived from Ziziphus jujube (fruit or seeds), and its bioavailability and druglikeness is much better than C-glycosides and jujubosides. These findings recommend that the involvement of zizyphusine in quality control of SZJ extract and pharmacologic actions in anxiolytic effect is worth investigating in the future. Because the pharmacologic study of zizyphusine is poor at present, more attention could be paid to research and development of zizyphusine as a potential natural anti-anxiety drug.

There are some limitations of our current research. First, we did not conduct the behavioral test to confirm the anxiolytic-like effect of SZJ. Such benefit of SZJ was concluded on the base of previous pharmacodynamic studies, as well as clinical practice experience of traditional medicine. Our in vitro mRNA expression evaluation of GABA_A and 5-HT receptors only used a single concentration of SZJ extract, which corresponds to approximately 90% cell viability in CellTiter-Glo assay. Leveraging the integrated approach of system biology, UPLC-Q-TOF/MS and RT-qPCR, present work contributed to the illustration of potential mechanism of action involved in the anxiolytic-like effect of SZJ. However, further in-depth preclinical studies are warranted to verify the results obtained from the current analysis.

In conclusion, our results systematically demonstrated that anxiolytic mechanisms of SZJ mainly involved the regulation of serotonergic and GABAergic synapse pathways, in which the two-way modulation of GABRA1, HTR1A, and HTR2A may play an important role. In addition to C-glycosides and jujubosides, triterpenic acids and zizyphusine identified in SZJ also contributed to the regulation of serotonergic and GABAergic synapse pathways. This study provides directional predictions of anxiolytic mechanism of SZJ and insights to improve the quality control of standard extraction.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

LC, XZ, and JK designed the study. XZ, YZ, LZ, and BL performed the experiments. LC, CH, and JD prepared the manuscript. All authors contributed to the article and approved the submitted version.

The Nutrilite Health Institute fully funded this study.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.