OBJECTIVE To investigate the material basis and mechanism of the intestinal toxicity of Euphorbia ebracteolata.

METHODS Different extract fractions of Euphorbia ebracteolata were orally administered to normal mice. The toxic fraction was screened based on the release levels of inflammatory cytokines TNF-α and IL-1β in ileal tissue, changes in gene and protein expression of tight junction proteins ZO-1, Claudin-1, and Occludin, serum levels of intestinal barrier injury markers DAO and D-LA, and histopathological examination of intestinal tissue. The toxic fraction (dichloromethane extract) was further separated by silica gel column chromatography. Toxic components were screened in vitro by monitoring changes in mRNA levels of tight junction proteins (ZO-1, Claudin-1, Occludin) in IEC-6 cells and mRNA levels of pro-inflammatory cytokines (TNF-α, IL-1β) in RAW264.7 cells. The chemical composition of the toxic subfraction was analyzed by LC-MS/MS, and the major terpenoids identified were further validated for toxicity in vitro.

RESULTS The dichloromethane fraction of Euphorbia ebracteolate induced pathological injury in mouse ileal tissue, significantly increased the release levels of TNF-α and IL-1β in ileal tissue and serum levels of DAO and D-LA (P<0.01), and significantly decreased the gene and protein expression levels of ZO-1, Claudin-1, and Occludin (P<0.05, P<0.01). Subfraction Fr.6 from the dichloromethane fraction exhibited pro-inflammatory and intestinal barrier-damaging effects. UPLC-MS/MS analysis indicated that Fr.6 mainly contained diterpenoids. Six major diterpenoids isolated from Fr.6 were evaluated for toxicity in vitro. The results showed that Euphorin G (EG) and Ebraphenol C (EC) significantly decreased the mRNA expression of tight junction proteins in IEC-6 cells (P<0.05, P<0.01) and significantly increased the permeability of intestinal organoids (P<0.05, P<0.01). Jolkinolide B (JNB), Fischeria A (FA), and 11β-Hydroxy-8,14-epoxy-ent-abieta-13(15)-en-16,12β-olide (HEA) significantly increased the mRNA expression of TNF-α and IL-1β in RAW264.7 cells (P<0.05, P<0.01).

CONCLUSION The dichloromethane fraction of E. ebracteolate induces intestinal toxicity by disrupting the intestinal barrier and causing intestinal inflammation. The primary material basis for this toxicity is diterpenoids.