The results of OF test indicated that BEO significantly prolonged the time spent in the central area by approximately 0.52-fold (Fig. 1). Studies have shown that β-caryophyllene gavage in mice at a dose of 200 mg/kg also significantly prolongs the time spent in the central area by approximately 0.34-fold [10]. However, the actual concentration of β-caryophyllene in this study was only 64 mg/kg (Table S1), thus indicating that the active components in BEO are derived from β-caryophyllene. For example, intra-dorsal hippocampal injection of pure borneol [6] has anxiolytic effects, thereby indicating that borneol is also an active anxiolytic compound found in BEO.
The results of EPM test indicated that BEO prolonged the time spent in the open arms by approximately 1.2-fold and increased the number of entries on open arms by approximately 1.5-fold (Fig. 2) that in the control group. The β-caryophyllene used in the OF test also significantly prolonged the time spent on the open arms by approximately 0.6-fold when the mice were intragastrically administered a dose of 200 mg/kg. The number of entries into open arms significantly increased by approximately 0.4-fold [10], in agreement with previous results showing that the active components of BEO are derived not only from β-caryophyllene but also from other components. According to preliminary experiments (Table S1), the components of BEO, in addition to β-caryophyllene (10.6%), borneol (16.4%), and limonene (8.2%), have been tested with the EPM and demonstrated to possess anxiolytic effects [6, 8]. The above results indicate that β-caryophyllene, limonene, and borneol are the active components involved in the anxiolytic effect of BEO.
The results of LDB test indicated that BEO treatment (600 mg/kg), compared with the control group, significantly prolonged the light area duration by approximately 0.45-fold and increased the transition number by approximately 0.67-fold (Fig. 3). The β-caryophyllene described above also prolonged the light area duration by approximately 0.47-fold at a dose of 200 mg/kg and increased the transition number by 0.4-fold [10]. The actual concentration of β-caryophyllene found in BEO in this study was only 64 mg/kg (Table S1), thereby indicating that other components in BEO and β-caryophyllene have synergistic effects.
Further network pharmacological analysis results shown that CNR2 is associated with anxiety and depression [7], and AR has been confirmed to be involved in brain function and the regulation of anxiety [17]. ESR1 has also been confirmed to be associated with anxiety and to be a cause of frequent anxiety in women [18]. Furthermore, low ACHE activity has been associated with an increased risk of depression and anxiety in adolescents [19], and SLC6A4 is also associated with anxiety and depression [20]. Selective serotonin reuptake inhibitors are the main treatments for major depression and anxiety, and CYP2C19 polymorphisms are known to affect the metabolism of these drugs, thereby affecting their efficacy and safety [21].
In addition, studies have shown that borneol acts on the transient receptor potential cation channel vanilloid subfamily member 1 (TRPV1) and exerts analgesic effects [22]. Moreover, α-pinene, β-pinene, camphor, and limonene inhibit ACHE activity, and limonene also inhibits BCHE activity [19], whereas β-caryophyllene acts on CNR2, thereby producing its anxiolytic effect [7]. These results show that these components and targets are important for the regulation of anxiety with BEO.
Based on analysis of the KEGG signaling pathway (Fig. 4B), among them, the neuroactive ligand-receptor interaction signaling pathway controls and regulates many important biological functions such as emotion, memory, and endocrine function [23]. Anxiety and metabolic disorders are closely related, and both have common pathological manifestations, such as chronic inflammation [24]; moreover, long-term stress and anxiety can induce tumors and promote the development of cancer [25]. Both depression and anxiety are early manifestations of neurodegenerative diseases and Alzheimer's disease [26]. In addition, the serotonergic synapse pathway is associated with the occurrence of anxiety [23]. The cAMP signaling pathway also has anxiolytic effects, through regulating intracellular cAMP levels [27]. Therefore, BEO regulates anxiety through multiple signaling pathways, thus underscoring its multi-component, multi-target, and multi-pathway function. Furthermore, our current research is essentially consistent with previous results and thus should have value for theoretical guidance. Based on the analysis of GO enrichment analysis (Fig. 5A), among these, G-protein coupled receptor signaling pathways are involved in the development of anxiety disorders and may be a potential therapeutic target [28]. The steroid hormone mediated signaling pathway has also been confirmed to be associated with anxiety [29]. Interestingly, the sedative and hypnotic mechanism of action of a compound found in Anshen essential oil is similar to those of components in BEO and are also associated with calcium ion transport into the cytosol, response to ammonium ions, and RNA polymerase II transcription factor activity [13]. The above studies are essentially consistent with the findings of this study and indicate that BEO regulates anxiety through modulating ion homeostasis, transcriptional regulation, and signal transduction. Among the result of PPI network, AR and ESR1 are associated with a variety of human behaviors, including anxiety [30]. PPARG has been confirmed to be associated with pathological anxiety [31]. glucocorticoid receptor (NR3C1) [32] and HTR2A [33] are also associated with anxiety disorders. An estrogen-dependent interaction between ESR1 and EP300[34] and an interaction between NR3C1 and EP300 in transcription activation[35] have been reported. ADORA2A and D2D2 have been shown to have a synergistic effect on anxiety disorders [36]. The above results are consistent with those of this study. Subsequently, we further focused on the potential therapeutic targets of anxiety disorders and used molecular docking analysis of the active components and potential targets obtained from the screening.
Based on analysis of the molecular docking, among these, ADRA2B [37] and ADORA2A [36] have been reported to be closely associated with anxiety. In addition, camphor, α-pinene, linalool, and methyl eugenol have high affinity for EP300, MIF, and leucine rich repeat kinase 2 (LRRK2) targets. Furthermore, EP300 [38], MIF [39], and LRRK2 [40] have been confirmed to be associated with anxiety (Fig. S1), thus indicating that the above components and targets are significantly related to anxiety and are worthy of further investigation. This study determined and confirmed that the main active components in BEO, including borneol, β-caryophyllene, α-cadinol, limonene, and α-selinene, act on key targets, such as CNR2, ADRA2B, and ADORA2A. This study therefore provides new avenues for further research on the pharmacodynamic basis of BEO for the treatment of anxiety. Moreover, it should promote future basic research to determine novel targeted drugs for the treatment of anxiety disorders.