Mechanisms Underlying the Action of Ziziphi Spinosae Semen in the Treatment of Insomnia: A Study Involving Network Pharmacology and Experimental Validation

Abstract

Purpose: This study aimed to investigate the potential mechanisms and related bioactive components of ZSS for the treatment of insomnia.Method: The insomnia model of rat induced by PCPA was established. After oral administration of ZSS extract, the general morphological observation, pentobarbital sodium-induced sleep test and histopathological evaluation were carried out. Network pharmacology, assisted by UHPLC-Q-Exactive-MS/MS analysis, was developed to identify the targets of ZSS in the treatment of insomnia, as well as the corresponding signaling pathways. In addition, we validated the identified targets and pathways by RT-qPCR and immunohistochemical analysis.Results: The pentobarbital sodium-induced sleep test, determination of 5-HT and GABA levles in hypothalamic tissues and HE staining showed that ZSS extract was an effective treatment for insomnia. Network pharmacology analysis identified a total of 19 candidate bioactive ingredients in ZSS extract, along with 433 potentially related targets. Next, we performed protein-protein interaction (PPI), MCODE clustering analysis, GO functional enrichment analysis, KEGG pathway enrichment analysis, and ingredient-target-pathway (I-T-P) sub-networks analysis. These methods allowed us to investigate the synergistic therapeutic effects of crucial pathways, including the serotonergic and GABAergic synapse pathways. Our analyses revealed that palmitic acid, coclaurine, jujuboside A, N-nornuciferine, caaverine, magnoflorine, jujuboside B, and betulinic acid, all played key roles in the regulation of these crucial pathways. Finally, we used the PCPA-induced insomnia in rats to validate the data generated by network pharmacology; these in vivo experiments clearly showed that pathways associated with the serotonergic and GABAergic system were activated in the rats model. Furthermore, ZSS treatment significantly suppressed high levels of HTR1A, GABRA1, and GABRG2 expression in the hypothalamus and reduced the expression levels of HTR2A.Conclusion: Based on the combination of comprehensive network pharmacology and in vivo experiments, we successfully identified the potential pharmacological mechanisms underlying the action of ZSS in the treatment of insomnia. The results provide a theoretical basis for further development and utilization of ZSS, and also provide support for the development of innovative drugs for the treatment of insomnia.

Structure revision of the aporphine zizyphusine alkaloid from Semen Ziziphi Spinosae

“…Other aliphatic protons appeared in the region between δ 2.57 and 4.31. 13 C NMR and HSQC data revealed the presence of 20 carbon atoms, including nine sp 2 quaternary carbons, three sp 2 and one sp 3 CH, and three sp 3 CH 2 , which are characteristic signals of the aporphine alkaloid, [15] and the aporphine skeleton was confirmed by HMBC correlations from H-(6-NCH 3 , 2.87, 3H, s; 6-NCH3, 2.87, 3H, s) to C-5 (δ 60.4) and C-6a (δ 69.1); H-3 (δ 6.61, 1H, s) to C-4 (δ 23.2); H-8 (δ 6.38, 1H, d) to C-7 (δ 30.4). The HMBC correlations from H-(2-OCH 3 , δ 3.68, 3H, s) to C-2 (δ 151.1) and C-3 (δ 108.7) attached one methoxy group to C-2.…”

“…The majority of the literature either refers to Han et al's article [4] or reported MS data only. Moreover, many of the articles claimed there was magnoflorine present in the Semen Ziziphi Spinosae, [12][13][14] which is remarkably similar to zizyphusine, and it was also proposed to be one of the potential bioactive components for the treatment of insomnia. [13] In view of this situation, the structure of zizyphusine was reexamined.…”

“…The chemical structure of zizyphusine was revised by systematic analyses of high resolution electrospray ionization-time of flight mass spectrometry (HR ESI-TOF MS), 1 H, 13 C NMR, 1 H-1 H correlation spectroscopy (COSY), heteronuclear singular quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC) spectra. In addition, in order to contrast the NMR spectral data of the acetylate proposed by the article, acetylation was also performed.…”

Integrating animal experiment, mass spectrometry and network-based approach to reveal the improving sleep quality of Ziziphi Spinosae Semen and γ-aminobutyric acid mixture

“…Until now, the pharmacological investigation of ZSS for the treatment of insomnia has mainly focused on rat models. For instance, extensive studies from para-chlorophenylalanine-induced insomnia model rats demonstrated that ZSS aqueous extract (30 g/kg) or ZSS alcohol extract (403.38 mg/kg) signi cantly enhanced the serum concentration of 5-TH and enhanced sleeping behaviors, indicating that the extract was effective in treating insomnia in rats [38,39]. Another main ingredient GABA, one of the major inhibitory neurotransmitters in the nervous system, generates a neuroinhibitory effect by binding with its receptor.…”

Shumian Capsule Improves the Sleep Disorder and Mental Symptoms Through Melatonin Receptors in Sleep-Deprived Mice

“…Sleep latency was defined as the time between the injection of pentobarbital sodium and the beginning of losing righting reflex loss. Sleep duration was defined as the time from the beginning to the recovery of righting reflex loss (Bian et al, 2021).…”