Exploring the antioxidant activity and mechanism of wild silkworm soybean root based on network pharmacology and in vitro experiments
Wild silkworm bean root is the root of Centrantheragrandiflora Benth, a plant in the Scrophulariaceae family, mainly distributed in Yunnan, Guizhou, Guangxi and other places. Wild silkworm bean roots are medicinal and edible plants, rich in various active substances such as cyclohexene ether terpenes, phenylethanolic glycosides, ionones, carotenoids, monoterpenes, and other compounds. They have physiological activities such as antioxidant, anti-inflammatory, anticoagulant, and myocardial ischemia, as well as promoting wound healing. Ancient books record that it is used to make wine to prevent and treat diseases such as cerebral thrombosis and stroke. It is planted as Yi ethnic medicinal herbs in Yunnan and widely used in ethnic medicine. At present, research mainly focuses on the extraction and separation of wild silkworm bean roots, as well as activity studies. Wild silkworm bean roots are rich in iridoid glycosides, which have antioxidant, lipid-lowering, hepatoprotective, and preventive effects against cardiovascular and cerebrovascular diseases, and have great potential for development.

Oxidative stress refers to a stress state in which the body is stimulated to produce excessive reactive oxygen species (ROS), leading to an imbalance with antioxidants and causing cardiovascular disease, neurological disorders, and cancer. Due to the potential harm of processed and synthesized antioxidants to health, people are more committed to seeking natural antioxidants. Natural antioxidants are increasingly attracting attention due to their significant therapeutic effects and low toxicity and side effects. Wild silkworm bean root, as a natural medicine, has good antioxidant activity, but there is limited research on its specific active ingredients and mechanism of action.

Network pharmacology is an emerging discipline that reveals the regulatory network of drugs in the body at the systemic level. By constructing a complex network relationship between drugs, active ingredients, targets, and diseases, the mechanism of action of drugs can be predicted, especially for the prediction of the mechanism of action of multi-target drugs in traditional Chinese medicine development. This article uses network pharmacology to analyze the main active ingredients and target components of wild silkworm soybean root, and deeply explores the molecular mechanism of its antioxidant effect. It is verified through in vitro antioxidant experiments and biological methods, providing a theoretical basis for further basic experimental research on wild silkworm soybean root.

Wild silkworm bean root
Wild silkworm bean root is a traditional Chinese medicine in China, which contains various active substances such as cyclohexene ether terpenes, phenylethanol, carotenoids, etc. It has various pharmacological activities such as antioxidant, hypoglycemic, anticoagulant, and myocardial ischemia. At present, research on the antioxidant activity of wild silkworm bean roots is gradually deepening, but the specific active ingredients and mechanisms have not yet been discovered. In order to further explore the mechanism of antioxidant stress of wild silkworm soybean root, this study conducted in vitro antioxidant experiments, network pharmacology, molecular docking technology, and Western blot detection to further investigate it.
Through in vitro antioxidant experiments, it was found that wild silkworm bean root extract has the ability to scavenge DPPH free radicals, ABTS free radicals, and hydroxyl free radicals, and within a certain concentration range, the three free radical scavenging rates of wild silkworm bean root extract have a certain linear relationship. At a concentration of 320 μ g/mL, the clearance rates of the three free radicals were 3.86%, 83.89%, and 59.15%, respectively. The IC50 values were 0.166mg/mL, 0.054mg/mL, and 0.180mg/mL, respectively, indicating good antioxidant capacity.
Through literature review and relevant database screening, 22 active ingredients of wild silkworm soybean roots were obtained, including aucubin, rhodopsin, β – sitosterol, geniposide methyl ester, catalpol, etc. Among them, aucubin in peach leaves has the highest content in wild silkworm bean roots and has a wide range of activities such as antioxidant, antibacterial, and anticancer. Nrf2 is a core transcription factor for antioxidant stress, regulating the activation and expression of downstream antioxidant factors and maintaining cellular homeostasis. Studies have found that aucubin can inhibit the differentiation of mouse osteoclasts through Nrf2 mediated antioxidant pathways, thereby slowing down the development of osteoporosis. Rhododendron red pigment is a natural carotenoid and an important active ingredient in wild silkworm bean roots. Research has found that rhodopsin in wild silkworm bean roots can exert a protective effect against cellular oxidative stress damage by increasing the activity of superoxide dismutase (SOD). Meanwhile, it can also exert protective activity against myocardial injury by stimulating the Nrf2 ARE pathway. Zichun is also a type of iridoid glycoside compound with a wide range of pharmacological activities such as anti-inflammatory and antioxidant properties. Research has found that Zichun can also upregulate the expression of Nrf2 and HO-1, increase SOD activity, reduce MDA activity, decrease brain cell apoptosis, promote Bcl-2 protein expression, inhibit Bax protein expression, and alleviate oxidative stress damage.
In protein interaction network analysis, 82 targets of antioxidant stress of active ingredients from wild silkworm soybean roots were identified. It mainly includes 23 core targets such as STAT3, HSP90AA1, HDAC1, PPARG, HSPAB1, etc. Heat shock proteins (HSPs) are molecular chaperone proteins that can alter the structure and interactions of other proteins and are induced by heat shock and other chemical and physical stresses. HSP90AA1 and HSP90AB1 belong to the HSP90 family of HSPs. When oxidative stress occurs, it activates the expression of intracellular heat shock proteins. Research has found that HSP90 can regulate oxidative damage in the body by modulating the Keap1 (INrf2) – Nrf2 signaling pathway. Under oxidative stress, HSP90 reacts with Keap1, promoting the dissociation of Keap1 Nrf2 complex and releasing Nrf2. Protecting cells from oxidative stress through transcriptional activation of Nrf2 and its downstream proteins. STAT3 is an important mediator of cell survival after oxidative stress, with strong proliferative and apoptotic resistance effects. Research has shown that STATA3 can exert its cellular protective effect by upregulating the expression of anti apoptotic protein Bcl XL and promoting the inactivation of caspases. HDAC1 is a type of enzyme that catalyzes the deacetylation of histones and non histones, and can reduce the expression of antioxidant enzymes through the deacetylation of histones and transcription factors. Meanwhile, studies have found that inhibiting the expression of HDAC1 can prevent oxidative stress imbalance induced by chemical hypoxia in H9c2 cells by improving cell viability and increasing SOD activity.
KEGG enrichment analysis showed that the antioxidant capacity of wild silkworm soybean roots is mainly related to the PI3K/Akt signaling pathway, nucleotide metabolism pathway, estrogen signaling pathway, etc. The PI3K/Akt signaling pathway plays an important role in antioxidant stress and is widely present in mammalian cells with good neuroprotective properties. The activation of the PI3K/Akt signaling pathway will promote the phosphorylation of Akt cascade and prevent oxidative damage. Research has shown that regulating the PI3K/Akt signaling pathway can improve apoptosis and oxidative stress in chondrocytes and H9c2 cardiomyocytes.
The molecular docking results showed that there was a good binding effect between the active ingredients of wild silkworm bean root and the core target, with strong mutual binding ability and close interaction.
PI3K belongs to cytoplasmic lipid kinases. When PI3K is phosphorylated, it recruits and induces Akt phosphorylation, thereby regulating physiological activities such as oxidative stress and apoptosis. Western blot analysis showed that the n-butanol extract of wild silkworm soybean root can promote the phosphorylation of protein PI3K and Akt, thereby activating the PI3K/Akt signaling pathway and improving oxidative stress.
In summary, this article evaluated the antioxidant activity of wild silkworm soybean roots using in vitro antioxidant experiments. The active ingredients, targets, and signaling pathways of antioxidant stress in wild silkworm soybean roots were predicted using network pharmacology and molecular docking techniques. Finally, Western blot analysis was used to verify its mechanism of action. The results showed that the IC50 values of wild silkworm bean root extract for scavenging DPPH free radicals, ABTS free radicals, and hydroxyl free radicals were 0.166, 0.054, and 0.180 mg/mL, respectively, confirming its good antioxidant activity. The results of network pharmacology show that wild silkworm bean roots can improve oxidative stress through multiple components, targets, and pathways, among which the PI3K/Akt signaling pathway may be the key pathway for its antioxidant effect. Western blot analysis further revealed that the extract of wild soybean root can activate the PI3K/Akt signaling pathway by promoting the phosphorylation of related proteins, thereby exerting antioxidant activity. This validates the mechanism of action of wild soybean root against oxidative stress, providing direction and laying a foundation for further research on the antioxidant stress effect of wild soybean root.