Three New Compounds from the Endophytic Fungus Trichoderma oligosporum in Croton tiglium, with Anti-Inflammatory Activity and Molecular Dynamics Studies

Background: Endophytic fungi are prolific sources of structurally diverse and biologically active compounds. However, anti-inflammatory constituents of Trichoderma oligosporum, an endophyte of the medicinal plant Croton tiglium, remain largely unexplored. This study aimed to isolate and characterize anti-inflammatory compounds from this fungus. Methods: Compounds were isolated from T. oligosporum and characterized by 1D/2D NMR and HRMS. Nitric oxide (NO) production was measured in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, with dexamethasone as control; cytotoxicity was assessed by CCK-8 assay. Compound 2 was further studied by ELISA, qRT-PCR, and Western blotting, and potential targets were probed by molecular docking and dynamics simulations. No preregistration was required, as no humans or animals were studied. Results: Three new compounds, oligosporins D–F (1–3), and 17 known compounds (4–20) were isolated. Compounds 1–3 inhibited NO production with IC50 values of 40.05 ± 4.92, 30.56 ± 4.74, and 51.88 ± 4.69 µM (dexamethasone, 40.43 ± 3.59 µM), while 14 and 19 were less potent; none was cytotoxic. Compound 2 reduced pro-inflammatory mediator secretion and down-regulated iNOS, TNF-α, and IL-6 mRNA. It also attenuated LPS-induced phosphorylation of IκBα and NF-κB p65 and restored IκBα expression. In all simulations, compound 2 remained bound within or near the iNOS heme pocket (MM-GBSA, −16.77 to −27.64 kcal/mol). Conclusions: Oligosporins D–F (1–3) are new compounds from T. oligosporum, with compound 2 being the most promising anti-inflammatory candidate. It apparently acts at least partly by inhibiting iNOS-mediated NO production and NF-κB activation. These findings warrant further investigation of T. oligosporum as an anti-inflammatory source.