Revealing the anti-ferroptosis guanidine alkaloids from Buthus martensii Karsch via a NMR-guided isolation combined with in vitro, in silico, and network pharmacology

By combining NMR fingerprint with rapid activity screening of eight crude fractions, eleven guanidine alkaloids were obtained from Buthus martensii Karsch, including one new natural product 7 and one new compound 10. Fraction 6 (IC50 = 141.40 ± 0.21 μg/mL) and Fraction 7 (IC50 = 50.89 ± 1.43 μg/mL) showed radical scavenging activity, and compound 8 exhibited the most potent free radical scavenging activity with an IC50 value of 5.09 ± 0.12 μg/mL. Their anti-ferroptotic capacities were validated across FerroOrange staining, which revealed that compounds 1 and 7 significantly inhibited erastin-induced ferroptosis in H9c2 cardiomyocytes. Through network pharmacology, these alkaloids appeared to exert their effects by modulating core targets related to ferroptosis, such as AKT1, EGFR, SRC, HSP90AA1, CASP3, and ESR1, with the notable impacts on the PI3K-Akt signalling pathway and Lipid and atherosclerosis. Molecular docking analysis confirmed the strong binding affinities and stabilities between guanidine alkaloids and these targets.