Fraxinellone

Dictamni Cortex (DC) has been associated with herb-induced liver injury, and fraxinellone (FRA), a furan-containing constituent, is implicated as a major hepatotoxic component. However, direct evidence linking FRA bioactivation to hepatic protein adduction in vivo remains limited. Here, we synthesized pyrroline- and pyrrole-type conjugates derived from the reactive FRA-cis-enedial intermediate and established liquid chromatography-tandem mass spectrometry methods to quantify corresponding lysine and cysteine/lysine adduct markers in the mouse liver after oral dosing with FRA or DC extract. Protein adduct levels increased in a dose- and time-dependent manner, peaked at 12 hours, and remained detectable up to 120 hours. Pretreatment with the CYP3A inhibitor ketoconazole markedly reduced adduct formation, supporting a CYP3A-dependent bioactivation pathway. To enable immunochemical detection, oxidized FRA was coupled to keyhole limpet hemocyanin to generate a polyclonal antiserum that selectively recognized FRA-derived pyrroline and pyrrole motifs. Western blotting and immunofluorescence revealed increased immunoreactive bands and a pericentral (central vein > portal triad) gradient in liver lobules, with prominent nuclear staining in hepatocytes after FRA or DC extract exposure; these signals were attenuated by ketoconazole. Collectively, these data demonstrate that CYP3A-mediated formation of an electrophilic cis-enedial intermediate drives covalent modification of hepatic proteins during FRA/DC exposure. The combined liquid chromatography-tandem mass spectrometry and antibody-based assays provide complementary tools for mechanistic studies and biomarker development for furan-containing herbal constituents. SIGNIFICANCE STATEMENT: This work provides direct in vivo evidence that CYP3A-dependent bioactivation of fraxinellone generates a reactive cis-enedial that covalently modifies hepatic proteins. The liquid chromatography-tandem mass spectrometry adduct markers and a selective antiadduct antibody enable semi‑quantitative detection and spatial mapping of protein adduction, supporting mechanistic investigations and biomarker development for furan-containing herbal constituents associated with liver injury.

Esophageal squamous cell carcinoma (ESCC), the predominant histological subtype constituting approximately 80% of all esophageal malignancies worldwide, represents a significant global disease burden with distinct geographical predilection. Fraxinellone (FRA) was reported to function as a tumor suppressor in several tumors, while the role of FRA in ESCC remains unclear. In the present study, we found that FRA treatment significantly inhibited cell proliferation and colony formation of ESCC cells. On the contrary, FRA treatment promoted cell apoptosis of ESCC cells by downregulating Bcl‐2 protein and upregulating Bax and C‐caspase3 proteins. Besides, FRA pretreatment suppressed proliferation and viability of CD8 + T cells but facilitated cell apoptosis in co‐culturing system. Moreover, FRA pretreatment restrained immune evasion by attenuating CD8 + T cell activation. Mechanistically, FRA effectively inactivated HIF‐1α/STAT3/PD‐L1 signaling in ESCC cells and xenograft tumors. Ultimately, FRA treatment inhibited tumor growth in vivo. In conclusions, FRA inhibited ESCC cell growth and immune evasion by inactivating HIF‐1α/STAT3/PD‐L1 signaling in vitro and vivo. This study suggested that FRA may serve as a potential therapeutic agent for ESCC treatment.