Butylphthalide

Inonotus hispidus (IH) is a species of traditional edible and medicinal fungus consumed in China, known for its anti-inflammatory, antioxidant, and anticancer pharmacological effects. IH has garnered widespread attention due to its therapeutic potential in clinical applications. However, despite its widespread use, comprehensive studies on its volatile chemical constituents and pharmacological mechanisms remain limited. In this study, the volatile components of IH at three growth stages (budding, mid-ripe, and full-ripe) were analyzed using headspace-gas chromatography-mass spectrometry (HS-GC-MS) and headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). Network pharmacology was further employed to investigate the mechanisms underlying its anti-inflammatory, antioxidant, and anticancer effects. Results showed that 20 and 37 volatile compounds were identified by HS-GC-MS and HS-GC-IMS, respectively, with alcohols and ketones being the predominant classes. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) revealed distinct separations among the three growth stages, with 16 key volatile organic compounds (VOCs) identified (VIP > 1). Relative odor activity value (ROAV) analysis highlighted 3-octanone, 3-methyl-1-pentanol, and 3-octanol as key flavor compounds contributing to earthy, mushroom, roasted, and grassy aromas. In the network pharmacology study, 34 compounds interacting with 59 common targets were identified. Protein-protein interaction (PPI) network analysis identified PTGS2, EGFR, ESR1, NFKB1, HIF1A, and STAT3 as core hubs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses elucidated the molecular mechanisms of IH's pharmacological effects. This study fills the research gap in IH's volatile components and deciphers their molecular mechanisms, providing a scientific foundation for quality control and further pharmacological investigations.

Sirtuin-2 (SIRT2) is an NAD⁺-dependent deacetylase that has been implicated in neuronal stress responses. The present study investigates the hypothesis that SIRT2 directly regulates LC3B acetylation under ischemic-like conditions, and that dl-3-n-butylphthalide (NBP) modulates this axis. In SH-SY5Y cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R), SIRT2 and the LC3B-II/I ratio increased, indicating autophagy activation. It was demonstrated that NBP was capable of restoring cell viability and normalizing SIRT2 and LC3B-II/I levels. Co-immunoprecipitation revealed the existence of an endogenous SIRT2-LC3B complex; SIRT2 overexpression resulted in a reduction of acetyl-LC3B, whereas the SIRT2 inhibitor AK-7 elevated LC3B-II/I. Molecular docking (AutoDock Vina) predicted stable NBP binding to the SIRT2 catalytic pocket, a prediction that was supported by 100-ns AMBER simulations (stable RMSD) and favorable binding free energy. The knockdown of SIRT2 resulted in the abrogation of the changes in LC3B-II/I that were driven by OGD/R- and NBP-, indicating a dependence on SIRT2. The findings of this study lend further support to the hypothesis that SIRT2 functions as an LC3B deacetylase under conditions of ischemic stress and suggest that NBP exerts its protective effect, at least in part, through the inhibition of SIRT2, thereby facilitating the normalization of autophagy.