Unveiling the hepatoprotective mechanisms of Desmodium heterocarpon (L.) DC: Novel flavonoid identification and Keap1/Nrf2 pathway activation

Background: The pathophysiology of liver diseases is significantly influenced by oxidative stress, making its alleviation a key strategy for treatment. The Keap1/Nrf2 signaling pathway is the body's most crucial antioxidant defense mechanism. Traditional Chinese medicine, Desmodium heterocarpon (L.) DC, has shown promising hepatoprotective effects, however, the specific active components and underlying mechanisms of its liver-protective properties remain inadequately understood. Further investigation into the bioactive constituents and mechanisms of its hepatoprotective action is therefore essential.

Objective: This study aims to identify the active ingredients in D. heterocarpon and to explore its hepatoprotective properties and underlying mechanisms.

Methods: The hepatoprotective activity of the ethyl acetate fraction (JEAE) from D. heterocarpon was first evaluated utilizing a mouse model of acute liver damage (ALI) caused by CCl₄. Molecular and histological analyses, including H&E staining, ELISA, and Western blot, were used to assess liver protection. The chemical constituents of JEAE were further identified using UPLC-MS/MS, and the molecular network of the JEAE fraction was analyzed. Compounds were isolated through column chromatography, and their antioxidant and hepatoprotective effects were assessed in an H₂O₂-induced HepG2 cell model using molecular assays. Additionally, binding interactions between active compounds and Keap1 were evaluated using molecular docking, molecular dynamics simulations, and surface plasmon resonance.

Results: The ethyl acetate fraction of Desmodium heterocarpon (JEAE) showed remarkable antioxidant activity, with the highest flavonoid contents among extract fractions. In CCl₄-induced liver injury models, JEAE improved liver function, reduced ALT and AST levels, and enhanced antioxidant enzyme activities, suggesting hepatoprotective effects via the Keap1/Nrf2 pathway. 47 compounds were identified in JEAE, and fourteen flavonoids, including two novel compounds (1 and 2), were isolated from the JEAE fraction. Compounds 1, 3, 5, 8, and 14 notably protected HepG2 cells from oxidative damage, reduced ROS levels, and maintained mitochondrial function. These compounds also showed strong binding affinities to Keap1 and other antioxidant receptors, with molecular dynamics simulations confirming their stability and binding potential as effective hepatoprotective agents.

Conclusion: This study demonstrates that the ethyl acetate fraction of Desmodium heterocarpon (JEAE) exhibits significant hepatoprotective effects, largely attributed to its flavonoid-rich composition. The protective effects are mediated through antioxidant pathways, particularly the Keap1/Nrf2 signaling pathway. Newly identified isoflavanes and other flavonoids in JEAE show strong potential as bioactive compounds, with stability and binding affinities supporting their role in reducing oxidative stress. These findings suggest D. heterocarpon as a promising source of hepatoprotective agents and provide a foundation for further exploration of its therapeutic applications.