Study on the mechanism of lemon essential oil in alleviating anxiety based on network pharmacology and experimental verification
Anxiety disorder is a neurological disorder characterized by temporary or sustained emotional tension as the main clinical symptom, accompanied by autonomic nervous system disorders, movement disorders, and other symptoms. It is also a mental illness characterized by unpleasant mood and physical discomfort. In recent years, the increasing pressure on people’s lives, studies, and work has led to a significant increase in the incidence of depression and anxiety disorders. According to statistics, the number of people suffering from depression worldwide accounts for 3.01% to 5.00%, and the number of people suffering from anxiety disorders accounts for 4.00% to 6.50%. At present, the main drugs used to treat depression and anxiety are Western medicines such as escitalopram, venlafaxine, and benzodiazepines. However, these drugs have significant side effects, strong dependence, and long medication cycles, which can cause certain harm to patients’ bodies.
Besides medication and psychological therapy, aromatherapy is considered an effective adjunctive treatment for anxiety disorders. Plant essential oils have a range of pharmacological properties, including anti anxiety, anti depression, sedative, neuroprotective effects, and other pharmacological activities. Many animal and human studies support the use of fragrances and their components to reduce anxiety related symptoms and behaviors. Plant essential oils such as bergamot, sweet orange, perilla, lemongrass, and lavender have a positive effect on improving anxiety symptoms through inhalation or massage methods without any adverse reactions. The linalool found in lavender essential oil and the geraniol found in rose essential oil are both effective in relieving anxiety. Research has found that lemon essential oil extracted from lemon peel can improve mood, cognition, relieve fatigue, and prevent cognitive decline during exam anxiety; Inhaling lemon essential oil (LEO) can significantly increase the beta waves of human brain waves. Martial et al. conducted behavioral experiments to study the anti stress effects of lemon essential oil vapor on mice and found that mice exposed to lemon essential oil had anti anxiety effects in both elevated maze and open field tests. Ze et al. studied the behavior, hormone, and neuronal responses of rats exposed to a diffuse lemon essential oil atmosphere. They found that rats spent less time in raising and maze opening arms, had an increased thermal injury threshold, and exhibited reduced formalin induced pain behavior. Corticosteroid concentrations in the hypothalamus and midbrain periaqueductal gray matter also decreased, indicating that LEO leads to significant changes in neuronal circuits related to anxiety and pain. However, LEO is a mixture of multiple components with the characteristics of “multiple components and multiple targets”, and its mechanism of anti anxiety has not been well elucidated.
Network pharmacology combines drug action networks with biological networks, based on the “drug target pathway disease” model, to analyze the interactions between drugs and organisms, and systematically explore the relationship between drugs and diseases. This study is based on network pharmacology methods to predict and analyze the key components, targets, and signaling pathways of lemon essential oil in anti anxiety. Combined with a series of experiments (antioxidant activity, cellular inflammation, and mouse anxiety model), the key components and targets in lemon essential oil screened by network pharmacology are verified, providing theoretical and experimental basis for the clinical application of lemon essential oil in anti anxiety.

This study uses network pharmacology methods to explore the key components, targets, and signaling pathways of lemon essential oil’s anti anxiety effects, in order to investigate the potential mechanism of lemon essential oil’s anti anxiety effects. By constructing a “lemon essential oil active ingredient target pathway anxiety” network, 19 key components (such as limonene, α – terpineol, methyl eugenol, β – pinene, orange blossom acetate, β – caryophyllene, 4-terpineol, etc.) and 18 key targets (such as GABRA1, GABRA2, GABRA5, MAO, SLC6A3, SLC6A2, DRD1, GRIA2, IL-6, etc.) were identified for the anti anxiety effects of lemon essential oil. The main pathways involved include neuroactive ligand receptor interactions, serotonergic synapses, dopaminergic synapses, nicotine addiction, cocaine addiction, and other signaling pathways.
The volatile compounds in lemon essential oil are mainly terpenoids, which have been found to have anti anxiety, depression, and cognitive improvement effects. Orange blossom ester acetate has anti-inflammatory and antioxidant effects, and is widely used in the treatment of conditions such as neuropathy and anxiety; By using a quantitative structure-activity relationship model to calculate the affinities for dopamine receptor D2, serotonin transporter, and 5-hydroxytryptamine receptor 1A, it was concluded that hesperetin acetate may be an effective antidepressant and neuroinhibitor. Orange blossom ester acetate can regulate monoamine oxidase A, prevent serotonin from generating 5-hydroxyindoleacetic acid under the action of monoamine oxidase A, reduce the concentration of serotonin in synaptic cleft, and thus alleviate anxiety.
DRD1 belongs to the metabolic G protein coupled receptor, which can reduce the activation of spinal cord neurons mediated by ionotropic glutamate receptors through G α q protein dependent inhibition of DRD1 through non receptor tyrosine kinase, thereby alleviating chronic pain. Studies have also shown that electroacupuncture stimulation of the lateral amygdala can activate DRD1 to alleviate nerve damage caused by anxiety behavior in mice. The subunit encoded by GRIA2 is highly expressed in neurons, and after knocking out GRIA2, mice showed reduced anxiety in elevated mazes. IL-6 is a cytokine associated with central nervous system neuroinflammation. Activation of the striatum in microglia upregulates the expression of inflammatory molecules, including IL-6, and induces negative emotions and loss of pleasure in mice.
Molecular docking confirmed that the core compounds of lemon essential oil, such as orange blossom ester acetate, limonene, β – pinene, 4-terpineol, and α – terpinene, have binding energies lower than -5 Kcal/mol with the core targets DRD1, GRIA2, and IL-6 proteins, indicating good binding activity. This suggests that lemon essential oil has a good molecular basis for anti anxiety disorders. This indicates that the anti anxiety mechanism of lemon essential oil is achieved through the mutual regulation of multiple components, targets, and pathways. Based on network prediction results, in vitro validation of lemon essential oil for anti anxiety in cells.
Once the body’s antioxidant mechanism is disrupted, oxidative stress can occur, leading to the production of inflammatory cytokines and promoting inflammation. Oxidative damage to DA neurons can lead to the occurrence of anxiety. Through the detection of DPPH and ABTS free radical scavenging, it was found that the scavenging effect of lemon essential oil was superior to that of monomeric orange blossom ester acetate. Meanwhile, lemon essential oil can reduce LPS induced ROS levels in HaCaT cells in a dose-dependent manner, effectively reducing inflammatory factors such as NO, TNF – α, and IL-6, thereby alleviating cellular inflammatory damage caused by oxidative stress. Lemon essential oil can also improve cognitive ability in mice during anxiety, alleviate tissue damage, and reduce serum levels of related proteins.
In summary, this study used network pharmacology methods to screen the main chemical components, targets, and signaling pathways of lemon essential oil intervention in anxiety disorders. Through experiments, it was verified that components such as limonene and orange blossom acetate in lemon essential oil regulate multiple signaling pathways by targeting DRD1, GRIA2, IL6, etc., reducing cellular oxidative stress and inhibiting neuroinflammatory responses, thereby intervening in anxiety disorders.
The research results indicate that compounds in lemon essential oil can regulate different targets, and the same target can intervene in different biological processes and signaling pathways, reflecting the characteristics of lemon essential oil’s multi pathway and multi target combined effects. However, due to the limitations of network pharmacology methodology and the complexity of chemical reactions that occur during the extraction of traditional Chinese medicine components, this study is mainly based on the results of bioinformatics and biochemical indicators. In the future, further improvements will be made from the perspectives of genomics or proteomics to verify and clarify the main regulatory targets and mechanisms of action of lemon essential oil.