Bioactivity-guided identification of flavonoids with cholinesterase and β-amyloid peptide aggregation inhibitory effects from the seeds of Millettia pachycarpa

Abstract Millettia pachycarpa Benth, a widely used anthelminthic drug in folk, is rich in flavonoids with various bioactivities. This study aimed to identify active flavonoids with anti-Alzheimer’s disease (AD) effect from its seeds by a bioassay-guided isolation. A novel rotenoid with unusual oxidative ring-opening skeleton ( 10 ) and nine known flavonoids ( 1 – 9 ) were obtained, and their structures were elucidated by NMR and HR-ESIMS analysis. Among all isolates, 7 and 8 showed selective butyrylcholinesterase (BChE) inhibitory activities (IC 50  = 2.34 and 11.49 μM, respectively), while 3 was classified as a dual-action inhibitor against acetylcholinesterase (AChE) and BChE (IC 50 AChE  = 17.14 μM, IC 50 BChE  = 5.68 μM). Further kinetic study revealed that 3 , 7 , and 8 were mixed-type BChE inhibitors, but 3 was a competitive AChE inhibitor. Their strong binding affinities to BChE were confirmed by fluorescence quenching analysis. Additionally, 3 and 8 exhibited potent inhibitory effects against β-amyloid peptide aggregation. These results revealed M. pachycarpa could be a valuable source for anti-AD leads development, and compounds 3 , 7 and 8 were worthy of further study as multifunctional or specific agents for AD treatment.