Exploring the mechanism of volatile components in Tibetan ephedra on bronchitis based on network pharmacology and molecular docking
Bronchitis is mainly caused by specific reactions of bronchial mucosa and surrounding tissues, with a long onset cycle. Its pathogenesis is mainly influenced by external factors such as seasonal changes, viral infections, allergic infections, as well as internal factors such as respiratory defense system and weakened immunity.

Ephedra, as a traditional Chinese medicine, has a long history of use. It has the effects of sweating, dispelling cold, promoting diuresis, reducing swelling, and clearing the lungs and asthma. It is a commonly used traditional Chinese medicine for bronchitis and contains multiple active ingredients. Research has found that flavonoids such as luteolin, apigenin, and quercetin in the combination of Ephedra sinica and Cinnamomum cassia have a therapeutic effect on bronchitis by targeting TNF – α and LTB4. However, there is currently no report on whether volatile components have an effect on bronchitis. Ephedra tibetana is a unique plant of Ephedra in Xizang, and its roots and stems have special aroma. The ancient Tibetan medical book “Jingzhu Bencao” records that “Caitun (ephedra) stops bleeding and clears spleen heat”, while “Zhonghua Tibetan Bencao” records “clears blood heat, heart heat, liver heat, spleen heat, new and old heat, promotes diuresis, stops bleeding, and relieves cough”. There have been reports on the volatile components of other ephedra plants, such as Ephedar sinica in Gansu, which mainly consists of d – α – terpineol (21.4%), hexadecanoic acid (14.74%), and 9-hexadecanoic acid (7.51%); The main volatile components of Shaanxi ephedra are 1- α – terpenol (28.57%) and 2,3,5,6-tetramethylpyrazine (7.92%); The main volatile components of Ephedra media are 1,4-eucalyptol (12.8%) and 1,8-eucalyptol (9.9%); The main volatile components of Ephedra equisetina are hexadecanoic acid (26.22%) and dibutyl phthalate (10.48%). Tibetan ephedra may have special medicinal substances due to its long-term adaptation to high-altitude environments. It has been found that the unique aroma of the roots and stems of Tibetan ephedra is different from that of traditional Chinese medicines such as Chinese ephedra and Chinese ephedra. What are the volatile components of ephedra, how do they differ from the volatile components of other ephedra plants, and how do they differ from the volatile components of ephedra roots and stems? There are currently no reports on this.
Using network pharmacology methods to screen active substances, predict target proteins, and search for signaling pathways can quickly predict the interactions between active ingredients, target proteins, and disease occurrence and development related pathway networks. Molecular docking is a drug design method that utilizes computer technology based on receptor characteristics and the interaction between receptors and drug molecules. Its main research basis is the conformational changes of ligands and receptor molecules during the binding process. Due to the lack of information on the chemical composition of Ephedra sinica in the online TCMSP database, GC-MS was used to infer the volatile components of Ephedra sinica. A small database was constructed based on TCMSP online data, and potential drug targets were queried using compound CAS numbers. Network pharmacology analysis and molecular docking were conducted to predict the mechanism of action of volatile components in Ephedra sinica for treating bronchitis.

This study found that the main volatile components in the stems of Tibetan ephedra are different from those reported in Chinese ephedra and Chinese ephedra. 2,3,5,6-tetramethylpyrazine is the main volatile component shared by both Tibetan ephedra and Chinese ephedra. In addition to ephedrine and pseudoephedrine, 2,3,5,6-tetramethylpyrazine is also the main active ingredient of ephedra and is widely used in food flavoring additives. The other two main components of the stem of Tibetan ephedra are Z-9-pentadecenol (9.15%) and dihydroxyacetophenone (7.92%). Z-9-pentadecenol has been reported as the main volatile component of Peucedanum praeruptorum stem, and its volatile oil has antibacterial activity against Escherichia coli, Salmonella typhi, and Shigella flexneri. However, whether Z-9-pentadecenol substance has activity has not been reported. Dihydroxyacetophenone has the effect of reducing mean pulmonary artery pressure in patients with chronic obstructive pulmonary disease, but its effect on bronchitis has not been reported. The main volatile components of Ephedra sinica roots are linoleic acid (7.81%), bisabolol (7.1%), and Z-9-pentadecenol (5.98%). Linoleic acid is an unsaturated fatty acid that has the effect of reducing blood lipids and softening blood vessels. Red myrrhol is the main component contained in plants such as Waldheimia glabra, Pogostemon specious, Eremantus erythropapus and Matricaria chamomilla in Xizang, and has anti-inflammatory, anti-cancer, antibacterial and antioxidant activities. The volatile components in the roots and stems of Tibetan ephedra, including palmitic acid, palmitic acid, and linoleic acid, are all unsaturated fatty acids with important nutritional and economic value. Alpha terpenol, linalool oxide, alpha limonene, thymol, and elephantol are also common components in plant volatile oils, with multiple activities such as antibacterial, anti-inflammatory, and antioxidant. Overall, it indicates that the volatile oil from the rhizome of Ephedra sinica has promising applications in food and drug development.

Bronchitis is mostly a sporadic disease with a relatively low prevalence. It often occurs during cold seasons or sudden climate changes, and is more likely to occur in the elderly and weak. Clinical validation has shown that ephedra has significant effects in treating acute bronchitis and pediatric bronchiolitis. Bronchitis is a disease with an inflammatory response mechanism. This study identified four core target proteins, namely IL6, TNF, PTGS2, and CXCL8, which may play an important role in the treatment of bronchitis with volatile components from Tibetan ephedra. IL-6 is a chemokine with multiple biological activities that can stimulate inflammation and autoimmune processes in various diseases. It has physiological characteristics such as activating and regulating immune cells. By participating in the differentiation of lymphocytes and monocytes, it promotes the maturation of B cells, secretes cytokines such as IgG, IgE, IgA, and participates in various inflammatory reactions and disease development processes. It is a drug target for treating infections, inflammation, autoimmune diseases, and cancer. TNF is a cytokine involved in systemic inflammation and is also one of the many cytokines that cause acute reactions. It is mainly secreted by macrophages and can induce cell death in certain tumor cell lines. TNF – α can activate neutrophils and macrophages to increase their cytotoxic effects, release more inflammatory cytokines such as IL-6 and IL-8, and accelerate the inflammatory response process. PTGS2 is responsible for producing inflammatory prostaglandins and is a lipid regulator that induces biological activity. Under the stimulation of inflammatory mediators, its level significantly increases, and the PTGS2 content in bronchoalveolar lavage fluid of asthma patients is higher, indicating that PTGS2 is associated with inflammation in asthma. CXCL8 is a cytokine secreted by macrophages and epithelial cells, which can attract neutrophils, eosinophils, and T cells, and also participate in the activation of neutrophils. It can be released from several types of cells to respond to inflammatory stimuli, and its main biological activity is to attract and activate neutrophils. Neutrophils undergo morphological changes upon contact with CXCL8, migrate directionally to the reaction site, and release a series of active products. These effects can lead to local inflammatory reactions in the body, achieving the purpose of sterilization and cell damage. In addition, CXCL8 also has certain effects on eosinophils, basophils, and lymphocytes.
This study found that 34 volatile components of Tibetan ephedra act on 32 targets of bronchitis. These targets include the main component of the stem, 2,3,5,6-tetramethylpyrazine, the main component of the root, linoleic acid, and the common components of the rhizome, benzaldehyde, α – terpineol, linalool oxide, 2,3,5,6-tetramethylpyrazine, geraniol, thymol, and elephantol. The targets that have good binding effects with IL6, TNF, PTGS2, and CXCL8 include eugenol, lauric acid, trans farnesol, α – terpineol, elephantol, and palmitic acid. Among them, eugenol and lauric acid are components of the roots, trans farnesol is a component of the stems, and α – terpineol, elephantol, and palmitic acid are common components of the roots and stems. From this, it can be seen that the volatile oils from the roots and stems of Ephedra sinica may have an effect in treating bronchitis. Dingzixiang phenol is considered a plant-based pesticide with fungicidal properties. Lauric acid has the effect of increasing the expression of anti-inflammatory factor IL-10 and reducing the expression of pro-inflammatory factor IL-6, which can alleviate the body’s inflammatory response to a certain extent. Farnesol has inhibitory effects on Streptococcus mutans, Lactobacillus acidophilus, Lactobacillus casei, and Actinobacteria naeslundii. High concentrations of farnesol inhibit the expression of IL-17 in mice infected with Candida albicans. Alpha terpenoids are present in plant volatile oils and have antibacterial, antioxidant, and anti-inflammatory effects. Their ester compounds have insecticidal effects. The above three substances all have the effect of inhibiting inflammatory factors, but palmitic acid has been reported to have a pro-inflammatory effect. Dingzixiang phenol and elephantol are common substances in plant essential oils, and their antibacterial effects have been reported to be significant along with other components in essential oils. This indicates that cinnamic acid, trans farnesol, α – terpineol, eugenol, and elephantol may participate in inflammatory reactions through their antibacterial effects. The GO enrichment results also showed that the targets of the above compounds were significantly enriched in the response process of bacterial derived molecules. Four target molecules are enriched together in the Toll like receptor signaling pathway. Toll like receptors belong to innate immune pathogen pattern recognition receptors, which can recognize invading pathogenic microorganisms and play a key role in inflammation, immune cell regulation, and other aspects. This indicates that eugenol, lauric acid, trans farnesol, α – terpineol, and elephantol in the volatile oil of the roots and stems of Ephedra sinica may inhibit the expression of IL6, TNF, PTGS2, and CXCL8 in inflammatory and immune signaling pathways such as TNF, IL-17, and Toll like receptors, thereby suppressing inflammation and immune responses of pathogenic microorganisms, and thus playing a therapeutic role in bronchitis. Palmitic acid triggers the secretion of pro-inflammatory cytokines, leading to an inflammatory response in the body.
This study is based on network pharmacology and molecular docking methods, taking the volatile components of the roots and stems of Ephedra sinica as the research object. The biological pathways and pathways of its intervention in bronchitis are comprehensively analyzed, and reverse molecular docking is used for verification. It is found that the intervention of active ingredients in bronchitis mainly presents the characteristics of multi molecules, multi targets, and multi pathways, and there is a synergistic effect among various volatile components. The research results provide reference for the experimental study and clinical application of Tibetan ephedra, but the results of this study only speculate the mechanism of the interaction between the volatile components of Tibetan ephedra and bronchitis through theory, and further pharmacological experiments are needed to verify it.