Metabolomics-guided identification of IFN-β modulating metabolites in Cladonia portentosa: effects of geographical origin and extraction methods

Natural products remain an important source of bioactive molecules. Factors such as environmental growth conditions influence the composition of secondary metabolites in marine and terrestrial species, while extraction methods can influence their yields and bioactivity. Metabolomics provides a powerful approach to investigate these factors and identify metabolites associated with bioactivity. Here, we combined 1 H NMR- and UHPLC-MS-based metabolomics with chemical and biological analyses to investigate the effects of sample origin (geographical location and extraction methods) on the chemical profile and immunomodulatory activity of Cladonia portentosa extracts. Six types of Cladonia samples collected from three Irish counties (Donegal, Kildare and Offaly), were extracted with methanol by conventional (Soxhlet) and green (ultrasonication and mechanochemical) extraction methods, yielding 18 different extracts. Extracts were chemically profiled by 1 H NMR while major metabolites were identified by UHPLC-MS and quantified by HPLC-DAD. Cell viability was assessed by the resazurin assay, and the immunomodulatory activity was evaluated in LPS-stimulated murine immortalised bone marrow derived macrophages (iBMDMs), measuring the pro-inflammatory cytokines, IL-6, TNF-α, IFN-β, and RANTES by ELISA. 1 H NMR based metabolomics differentiated Offaly samples from Donegal and Kildare samples based on the higher amounts of arabitol and perlatolic acid (PA), while UHPLC-MS identified usnic acid (UA), PA, olivetolic acid (OA) and 4- O -methylolivetolic acid as major secondary metabolites. The different extraction methods significantly impacted chemical composition as reflected in the cytotoxicity of the extracts in the order: mechanochemical > ultrasonication > Soxhlet. The extracts suppressed LPS-induced production of only IFN-β and IL-6. UHPLC-MS based metabolomics correlated IFN-β inhibition with PA, OA and 4- O -methylolivetolic acid in active samples. UA and PA exhibited cytotoxicity with IC 50 values of 48 ± 9 μM and 36 ± 4 μM respectively, whereas OA and 4- O -methylolivetolic acid showed immunomodulatory activity by suppressing LPS-induced IFN-β production. This study demonstrates the influence of geographical origin and extraction methodology on chemical composition and bioactivity of C. portentosa extracts. Although geographical origin affected the overall chemical profile, extraction methods were found to influence the composition and thus cytotoxicity. Importantly, these findings highlight the immunomodulatory potential of C. portentosa , contributing to the first report of IFN-β inhibition by 4- O -methylolivetolic acid and OA in iBMDMs.