Insomnia, characterized by difficulty falling asleep, shortened sleep time, and shallow sleep (Chen and Tang, 2021), often accompanies reduced memory and attention (Xu et al., 2019). Approximately one-third of the global population faces sleep problems, with 4–6% meeting clinical diagnostic criteria for insomnia (Chung et al., 2015). Prolonged insomnia can induce endocrine disorders, resulting in cognitive impairments, cardiovascular diseases, and other endocrine and immune disorders (Budhiraja et al., 2011; Javaheri and Redline, 2017), seriously impacting physical and mental health. In clinical practice, sedative-hypnotic drugs like benzodiazepine-receptor agonists, melatonin and variants, antidepressants, antipsychotics, and antihistamines are commonly prescribed for insomnia treatment (Cunnington et al., 2013). However, these drugs have limitations, including single-target action, drug resistance, addiction, withdrawal reactions, and limited efficacy against different insomnia types (Zhang and Xia, 2011). Traditional Chinese medicine (TCM) is gaining attention as a complementary and alternative therapy due to its advantages of minimal side effects, low risk of developing dependency, and ability to target multiple areas. TCM views insomnia as related to imbalances in Yin and Yang, disharmony between nutrient Qi and defensive Qi, and organ function imbalances. The holistic treatment approach involves techniques such as syndrome differentiation and treatment (Singh and Zhao, 2017). For instance, addressing restlessness stemming from stomach disharmony involves methods to harmonize the stomach and promote mental calmness, yielding favorable clinical outcomes (Zhang and Feng, 2014).

Ziziphi Spinosae Semen (ZSS) refers to the dried mature seeds of Ziziphus jujuba Mill., which belongs to the Rhamnaceae family. ZSS is recognized for its beneficial effects on the heart and liver. It nourishes these organs, calms the mind, and promotes body fluid production while reducing excessive sweating. ZSS is notably effective in addressing restless sleep caused by a deficiency of heart and liver blood, making it a frequent prescription for various insomnia types. Polygalae Radix (PR) designates the dried roots of either Polygala tenuifolia Willd. or Polygala sibirica L., which belongs to the Polygalaceae family. PR possesses diverse therapeutic properties, including mind-calming, cognitive function improvement, phlegm dissipation, expectoration promotion, and swelling reduction. Commonly used for insomnia, dreaminess, forgetfulness, and palpitations arising from heart and kidney imbalances, PR is a well-established sedative herb in Traditional Chinese Medicine (TCM). In clinical practice, PR is often combined with ZSS to treat insomnia, anxiety, and related conditions, with notable formulations such as Guipi Tang and Qifuyin. Some pharmacological data from animal studies, have demonstrated that ZSS possesses various pharmacological effects. These effects encompass sedative and hypnotic properties (Jiang et al., 2007), antidepressant activity (Li et al., 2022b), anxiolytic effects (Chen et al., 2023), and neuroprotection. The main active substances in ZSS, including flavonoids (e.g., spinosin), saponins (such as jujuboside A and jujuboside B), and alkaloids (e.g., magnolflorine), phenylpropanoids (e.g., coumestrol), and alkanoic acids are believed to underlie its sedative and hypnotic effects (Li et al., 2022c; Ren et al., 2023). Similarly, PR has demonstrated various pharmacological effects, such as memory improvement (Ren et al., 2022a), sedative and hypnotic properties (Sun et al., 2023), antidepressant activity (Chen et al., 2021), anxiolytic effects (Wen, 2006), and neuroprotection (Wang et al., 2023). The primary active ingredients in PR, such as polygala saponins, are deemed responsible for their sedative and calming effects (Cao et al., 2016). When combined with ZSS, PR exhibits a synergistic effect, further enhancing its sedative and hypnotic properties (Luo et al., 2020; Ren et al., 2022b). However, the components mentioned above in both ZSS and PR have limited content and bioavailability, presenting challenges in crossing the blood-brain barrier. Consequently, the specific mechanism of action and potential synergistic effects of Ziziphi Spinosae Semen and Polygalae Radix (ZSS-PR) in treating insomnia remain undetermined.

Accumulating evidence suggests a communication link between the brain and gut, termed the brain-gut axis. The gut microbiota can activate this axis, influencing neural, endocrine, and immune pathways that regulate physiological functions such as sleep, cognition, and disease progression (Li et al., 2020; Zhu et al., 2017). On one hand, the gut microbiota contributes to synthesizing neurotransmitters like 5-hydroxytryptamine (5-HT) and dopamine (DA). These neurotransmitters can directly or indirectly impact the central nervous system through the blood-brain barrier or vagus nerve, thereby regulating sleep. On the other hand, the interaction between the gut microbiota and the gut leads to the release of various cytokines and chemokines by gut secretory cells. These substances act on specific immune cells and the nervous system, thereby regulating sleep (Sun et al., 2016; Zhang et al., 2014). Conversely, insomnia can alter the structure and function of the gut microbiota through the brain-gut axis. Additionally, it has been observed that short-chain fatty acids (SCFAs) produced by gut microbiota serve as signalling molecules. These metabolites directly influence the central nervous system, gastrointestinal tract, and other peripheral nervous systems, thereby impacting the gastrointestinal and nervous systems through peripheral circulation. Metabolomics, a highly effective approach for systematically analyzing changes in endogenous metabolites due to external stimuli (Čuperlović-Culf, 2012), aligns with the research methods of TCM, emphasizing the study of holistic and multi-target mechanisms. Additionally, gut microbiota dysbiosis can worsen metabolic disorders, possibly arising from short-term sleep deprivation (Benedict et al., 2016). Previous research indicates that extracts from ZSS and PR significantly impact endogenous metabolic regulation (Duan et al., 2022; Heng et al., 2022; Luo et al., 2020). However, the precise mechanism by which ZSS-PR induces sedation and tranquillity via the mentioned neurological and endocrine indicators, along with its regulatory processes, remains unclear in the current academic literature.

This study aims to investigate the mechanism of ZSS-PR intervention in treating insomnia by utilizing metabolomics and gut microbiota analysis. Specifically, we examined the impact of ZSS-PR on the metabolic profile of animals with insomnia by UHPLC-HRMS metabolite profiling. Additionally, we assessed the influence of ZSS-PR on the composition and structure of gut microbiota in these animals through 16S rDNA high-throughput sequencing technology. The effect of ZSS-PR on the production of SCFAs in insomnia-affected animals was analyzed using gas chromatography-mass spectrometry (GC-MS). By integrating these findings, this study aims to unravel the intricate interrelationships among biochemical factors, metabolic profiles, gut microbiota, and SCFAs. Ultimately, our goal is to provide insights into the regulatory mechanism of the brain-gut axis and the therapeutic potential of ZSS-PR in managing insomnia.