Bioactivity and LC-HRMS Profiling of Methanol and Alkaloid Extracts from Fritillaria imperialis L. and Fritillaria pinardii Boiss. Bulbs

Fritillaria species are rich in steroidal alkaloids and are traditionally used for various medicinal purposes. This study investigated the biological activities of methanol, tertiary alkaloid, and quaternary alkaloid extracts obtained from the bulbs of Fritillaria imperialis and Fritillaria pinardii. Methanol, tertiary alkaloid, and quaternary alkaloid extracts prepared from the bulbs of both species were analyzed for secondary metabolites using LC-HRMS. Antimicrobial, cytotoxic, antioxidant, anti-inflammatory, and anticholinesterase activities were evaluated using standard in vitro assays. LC-HRMS analysis identified fumaric acid as the major metabolite in both species, together with (-)-epigallocatechin, cynarin, herniarin and several pyrrolizidine alkaloid N-oxides. Antibacterial activity was restricted to Staphylococcus aureus, whereas alkaloid-rich fractions displayed strong antifungal activity, particularly against Aspergillus fumigatus and Candida spp., with inhibition zones reaching 40 mm. At 80 μg/mL, the most pronounced reductions in cell viability were observed for F. imperialis methanol extract in A549 cells (55.19 ± 2.74%) and for F. pinardii tertiary alkaloid extract and F. imperialis quaternary alkaloid extract in MCF-7 cells (49.68 ± 1.83% and 49.76 ± 1.09%, respectively), whereas all extracts maintained J774 macrophage viability above 94%. Antioxidant activity was most pronounced in the quaternary alkaloid extract of F. pinardii (DPPH IC50 = 90.42 μg/mL; ABTS IC50 = 57.22 μg/mL), although all extracts were less active than the reference antioxidants. The tertiary alkaloid extract of F. pinardii exhibited potent anti-inflammatory activity, showing 5-LOX inhibition comparable to indomethacin (IC50 = 20.05 ± 0.23 μg/mL) and suppressing LPS-induced nitric oxide production by up to 62.22%. Among the tested samples, the tertiary alkaloid extract of F. pinardii exhibited the strongest cholinesterase inhibitory activity against AChE (IC50 = 13.39 ± 0.02 μg/mL). The results indicate that both F. imperialis and F. pinardii extracts exhibit measurable biological activities under in vitro conditions, with the alkaloid-rich extracts of F. pinardii showing comparatively stronger antifungal, anti-inflammatory, and anticholinesterase effects.