Exploring potential mechanism of ciwujia tablets for insomnia by UPLC-Q-TOF-MS/MS, network pharmacology, and experimental validation

Abstract

Insomnia, whether chronic or intermittent, is a common central nervous system disease. Ciwujia Tablet (CWT) is a well-known traditional Chinese medicine (TCM) made from the extract of Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. This medication is commonly used for treating insomnia in China, but the lack of in-depth research focused on the chemical ingredients of CWT creates a gap in knowledge regarding its effective constituents against insomnia. Considering that the therapeutic material basis, targets, and pathways related to this drug have not been fully investigated by scholars in the field, the focus of this study is on identifying the chemical ingredients or structural characteristics of CWT by the UPLC-Q-TOF-MS/MS technique. Besides, concepts of network pharmacology were also used to investigate the targets and pathways of CWT. An insomnia rat model was established by intraperitoneal injection of p-chlorophenylalanine, and the results were verified through various experiments. A total of 46 ingredients were identified in CWT, such as eleutheroside B, eleutheroside E, isofraxidin, and chlorogenic acid. Among them, 17 ingredients with good solubility, favorable gastrointestinal absorption, and high bioavailability were selected for network pharmacological analysis. It was concluded that CWT participated in the regulation of neurotransmitter levels, modulation of ion transport, neurotransmitter receptor activity, synaptic transmission, dopaminergic transmission and other essential processes. Results from the animal experiments showed that CWT can increase the content of inhibitory neurotransmitters 5-HT and GABA in the brain, reduce the synthesis of excitatory escalating transmitters DA and NE, shorten the sleep latency and prolong the sleep duration of insomnia rats. Furthermore, CWT could significantly alleviate the symptoms of insomnia in model rats. Identifying the chemical ingredients of CWT in this experiment is of great significance for exploring its potential curative effects, which provides a solid basis for further understanding the therapeutic value of this medication.

Keywords: UPLC-Q-TOF-MS/MS; ciwujia tablet; insomnia; nervous system disease; neurotransmitter.

FIGURE 1

Base peak chromatogram (BPC) of CWT test solution in the positive (A) and the negative ion modes (B).

FIGURE 2

MS/MS spectrum of chlorogenic acid in CWT and speculated fragmentation pathway.

FIGURE 3

MS/MS spectrum of eleutheroside B in CWT and speculated fragmentation pathway.

FIGURE 4

MS/MS spectrum of eleutheroside E in CWT and speculated fragmentation pathway.

FIGURE 5

MS/MS spectrum of isofraxidin in CWT and speculated fragmentation pathway.

FIGURE 6

Significant clusters analysis. (A): Cluster module (score = 12.30), including 21 nodes and 123 edges; (B): Cluster module (score = 11.38), including 14 nodes and 74 edges; (C): Cluster module (score = 5.33), including 7 nodes and 16 edges; (D): Cluster module (score = 4.00), including 14 nodes and 26 edges.

FIGURE 7

GO-BP, GO-MF, and GO-CC enrichment analysis diagram and KEGG pathway enrichment analysis diagram.

FIGURE 8

Sleep test results (C: control group, n = 10; M: model group, n = 10; E: estazolam group, n = 10; CWT-L group, n = 10; CWT-M group, n = 10; CWT-H group, n = 10; compared with the group C, *p < 0.05, **p < 0.01; compared with the group M, #p < 0.05, ##p < 0.01, mean ± SD). Figures (A,B): Test results on the fourth day of insomnia model establishment; Figures (C,D): Test results on the seventh day of treatment. (A): Detection of sleep latency of rats in groups C and M. (B): Detection of sleep duration of rats in groups C and M. (C): Detection of sleep latency of rats in each group. (D): Detection of sleep duration of rats in each group.

FIGURE 9

Neurotransmitter content in the brain (C: control group, n = 10; M: model group, n = 10; E: estazolam group, n = 10; CWT-L group, n = 10; CWT-M group, n = 10; CWT-H group, n = 10; compared with the group C, *p < 0.05, **p < 0.01; compared with the group M, #p < 0.05, ##p < 0.01, mean ± SD). Figures (A–D): Test results on the fourth day of insomnia model establishment; Figures (E–H): Test results on the seventh day of treatment. (A): 5-HT content analysis in groups C and M. (B): GABA content analysis in groups C and M. (C): NE content analysis in groups C and M. (D): DA content analysis in groups C and M. (E): Analysis of the 5-HT content in the brain of rats in each group. (F): Analysis of the GABA content in each group. (G): Analysis of the DA content in each group. (H): Analysis of the NE content in each group.