Research on the antioxidant damage of Codonopsis pilosula based on network pharmacology
Codonopsis pilosula (Franch.) Nannf. is a plant in the Campanulaceae family Codonopsis pilosula Nannf var. modesta (Nannf.) L. Dried roots of T. Shen or Codonopsis tangshen Oliv. Codonopsis pilosula is a commonly used tonifying traditional Chinese medicine in China, mainly containing carbohydrates, alkaloids, polyacetylenes, glycosides, terpenes and other ingredients. It has various pharmacological effects such as regulating blood sugar, promoting hematopoietic function, anti hypoxia, anti stress, anti fatigue, enhancing the body’s immunity, delaying aging, regulating gastric contraction, protecting gastrointestinal mucosa and anti ulcer. Up to now, there is a lack of research on characteristic active ingredients that are stable in nature, exclusive, and closely related to clinical efficacy in Codonopsis pilosula, which has become a bottleneck restricting the comprehensive quality control of Codonopsis pilosula.
Network pharmacology provides powerful methods and tools for the study of complex systems in traditional Chinese medicine. This method has been applied to interpret the compatibility rules of Chinese medicine formulas, screen active ingredients, study the basis of pharmacological substances, and investigate the mechanisms of action, achieving a series of research progress. However, current network pharmacology still faces some challenges, such as using network pharmacology to screen active ingredients for diseases that are not the main quality control components or components with higher content in traditional Chinese medicine according to current standards. Existing experimental evidence shows that some components obtained through network pharmacology screening have lower content in the original medicinal materials or compound formulas. So in order to find more valuable active ingredients of Codonopsis pilosula, this article searched for multiple components in Codonopsis pilosula that can be analyzed by HPLC through literature review. Using network pharmacology methods and “oxidative stress” as the search term, the main mechanism of action of multiple monomers in antioxidant stress was explored; And using H2O2 induced RAW264.7 cells as a model, the antioxidant effect of active ingredients in Codonopsis pilosula was verified, providing reference for the quality control of Codonopsis pilosula medicinal materials.

 

According to the prediction results of network pharmacology, the chemical formulas of Codonopsis pilosula alkynyl glycoside A and Codonopsis pilosula alkaloids B were not recorded in PubMed. Codonopsis pilosula glycoside I was not predicted as a target in Swiss Target Prediction, and there was one Codonopsis pilosula alkynyl glycoside Ning. The components that may have antioxidant effects among the 10 monomers are: Codonopsis pilosula alkynyl glycoside, Syringin, Codonopsis pilosula alkynyl glycoside B, L-tryptophan, Codonopsis pilosula alkynyl alcohol, and Codonopsis pilosula alkaloid A. And Codonopsis pilosula alkynyl glycoside, Codonopsis pilosula alkynyl glycoside Ning, Codonopsis pilosula alkynyl alcohol, and Heart leaf Codonopsis pilosula alkynyl glycoside B all belong to the Codonopsis pilosula alkynyl ene class components. Purple eugenol is a phenylpropanoid class component, and L-tryptophan and Guanhua Codonopsis pilosula alkaloid A are alkaloid class components. This article selects Codonopsis pilosula alkynyl glycosides, Syringin in phenylpropanoids, and L-tryptophan in alkaloids as representatives to conduct in vitro experimental studies on the antioxidant stress effects of the Nrf2 Keap1 signaling pathway. By constructing an in vitro H2O2 induced oxidative damage model in RAW264.7 cells, the protective effect on the model and its impact on the Nrf2 Keap1 pathway were investigated.

In the pharmacological effects of Codonopsis pilosula, there is a certain connection between anti stress, anti-aging, protection of gastrointestinal mucosa, anti fatigue and anti hypoxia and antioxidant pharmacological effects. Therefore, this article uses “oxidative stress” as the search term and adopts network pharmacology methods to explore the mechanism of Codonopsis pilosula’s antioxidant stress.

After intersecting the predicted target proteins of seven components of Codonopsis pilosula with oxidative stress-related proteins, 33 Codonopsis pilosula antioxidant damage target proteins were obtained. Construct protein-protein interaction networks for 33 intersecting targets, among which AKT1, CASP3, APP, MAPK1, MAPK8, MAPK3, and LCK are closely related. NFE2L2 and KEAP1 are important molecules in the Nrf2 Keap1 pathway, while APP, CASP3, CASP6, SYK, AKT1, GSK3B, MDM2, MMP2, PARP1, HSPA8, CDK2 can activate the Nrf2 signaling pathway by activating signaling pathways such as MAPK1/MAP2K1/MAPK3/MAPK9/MAPK8. Therefore, it is speculated that Nrf2 Keap1 may be an important pathway for potential active ingredients in Codonopsis pilosula to exert antioxidant damage effects. LCK, JAK2, EGFR, and other target proteins of Codonopsis pilosula active ingredients are closely related to immune inflammation, suggesting that Codonopsis pilosula may also exert antioxidant damage by regulating immune inflammation. GO enrichment analysis revealed 33 target proteins of potential active ingredients in Codonopsis pilosula for antioxidant stress, suggesting that these target proteins may exert antioxidant damage effects through molecular functions such as protein kinase activity, drug binding, nucleotide binding, as well as cellular response to oxygen-containing compounds and regulation of cellular biological processes.

In the pre experiment of the in vitro experiment, 490 μ mol/L of H2O2 was selected as the later intervention concentration, and it was found that 100 and 200 μ mol/L of Codonopsis pilosula acetylide, L-tryptophan, and syringin had no significant effect on RAW 264.7 cell proliferation after 24 hours of intervention. Therefore, 100 and 200 μ mol/L of Codonopsis pilosula acetylide, L-tryptophan, and syringin were used as experimental concentrations in the later stage.

He et al. determined the content of Codonopsis pilosula acetylide and Syringin in Codonopsis pilosula, which were 404.71 ± 5.90 and 25.23 ± 0.21 μ g/g, respectively. Gao measured the contents of Codonopsis pilosula acetylide, syringin, and L-tryptophan in dried, sun dried, and shade dried Codonopsis pilosula, which were 1134.7, 1581.3, and 1985.7 μ g/g for Codonopsis pilosula acetylide, 7.3, 27.6, and 121.6 μ g/g for syringin, and 96.9, 135.0, and 148.7 μ g/g for L-tryptophan, respectively. The content of Codonopsis pilosula alkynyl glycoside, Syringin, and L-tryptophan in Codonopsis pilosula can be quantified.

In summary, this article uses network pharmacology methods to predict the main mechanisms of antioxidant stress in the main potential active components of Codonopsis pilosula. Through cell experiments, the effects of representative components such as Codonopsis pilosula acetylide, L-tryptophan, and syringin on RAW264.7 cells treated with H2O2 were verified. The research results provide reference for the quality control of Codonopsis pilosula medicinal materials, decoction pieces, and preparations.