Pharmacological mechanisms of Nelumbinis plumula for insomnia: validation of key active ingredients and target pathways

Background Nelumbinis plumula (Lianzixin) is a traditional Chinese medicine with sedative and hypnotic properties. Our previous study discovered that natural bisbenzylisoquinoline alkaloids from Nelumbinis plumula exhibit good binding affinity to the orexin receptor (OXR), a target for insomnia treatment, through virtual screening. Herein, in this study, we evaluate the antagonistic activity of the principal bisbenzylisoquinoline alkaloids from Nelumbinis plumula on OXR and investigate the pharmacological effects and mechanisms in insomnia treatment. Methods Ca ²⁺ influx assays were used to detect the antagonistic effect of the principal bisbenzylisoquinoline alkaloids from Nelumbinis plumula on orexin receptors. The modified multi-platform method (MMPM) was used to establish chronic sleep deprivation mouse models. The open-field test (OFT) and pentobarbital sodium righting reflex test were used to observe the effect of NEF on insomnia. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) were used to explore the mechanisms involved in sleep-enhancing effects. Results The results of Ca ²⁺ influx assays found that neferine (NEF) had the strongest antagonistic effect among the main bisbenzylisoquinoline alkaloids from Nelumbinis plumula . Moreover, NEF significantly improved sleep quality and alleviated depressive-like behaviors in sleep-deprived mice. Mechanistically, orexin receptors are predominantly expressed in the locus coeruleus (LC) and dorsal raphe nucleus (DRN) brain regions, double immunofluorescence staining and neurotransmitters analysis showed that NEF inhibited the activities of noradrenergic and serotonergic neurons, leading to decreased norepinephrine (NE) and serotonin (5-HT) levels in sleep-deprived mice. Additionally, acute toxicity tests revealed that doses up to 2000 mg/kg of NEF exhibited no acute toxicity, indicating a high safety profile. Conclusion This work highlights that NEF is the primary active constituents of Nelumbinis plumula responsible for its anti-insomnia effects, with NEF can improve sleep quality and alleviate depressive-like symptoms by antagonizing orexin receptors and regulating noradrenergic and serotonergic neuron activities.