Sparalide B from Sparassis latifolia inhibits early adipogenesis in 3T3-L1 adipocytes via β-catenin-dependent downregulation of PPARγ and C/EBPα

Sparalide B, a phthalide isolated from Sparassis latifolia ( S. latifolia ), was structurally assigned as 4,5,7-trihydroxy-6-ethoxyphthalide by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR) ( 1 H, 13 C, heteronuclear multiple bond correlation (HMBC)) analyses. In 3T3-L1 adipocytes, sparalide B (2.5–20 μg/mL) significantly reduced lipid droplet accumulation in a dose-dependent manner, as quantified by Oil Red O staining at concentrations that did not affect cell viability. Western blotting showed decreased expression of adipogenic transcription factors (PPARγ, C/EBPα) and lipid droplet-associated/lipolytic proteins (perilipin-1, ATGL, HSL), alongside increased levels of thermogenesis/energy-metabolism markers (UCP2, PGC-1α, p-AMPK, AMPK). Stage-restricted treatments revealed that efficacy was confined to the early differentiation window (D0-D2), with little effect during D2-D4 or D4-D8. Mechanistically, sparalide B elevated β-catenin during D0-D2; β-catenin knockdown abrogated the compound's suppression of PPARγ/C/EBPα and its inhibition of lipid accumulation. These findings indicate that sparalide B targets early adipogenesis through β-catenin-dependent signaling to suppress lipid formation, highlighting this natural phthalide as a promising anti-obesity lead.