Namely the NOESY cross peaks between H
Namely, the NOESY cross-peaks between H-7″ methylene protons ( 4.01 and 4.16) with H-6′ ( 6.76) revealed that H-2 and OH-3 are -orientated, which indicated that the relative configuration of the C2 and C3 substituents as . For the -isomers, the thermodynamically more stable conformation is when H-2 and OH-3 are biaxial and therefore the absolute configuration has to be (2, 3). On the basis of above spectroscopic data, the structure of was elucidated and defined as a novel oxidative ring-opening rotenoid, named milletenone A. The inhibitory activities of all isolates on cholinesterases were evaluated by Ellman’s method and the results were summarized in . Most of isolates displayed moderate to weak AChE inhibitory effects with IC values ranging from 17.14 to 131.17 μM. By contrast, compounds , , and showed attractive BChE inhibitory activities with IC values ranging from 2.34 to 21.77 μM. It is well known that a decline in cholinergic activity in the Benzoylmesaconitine mg is closely associated with cognitive function loss of AD patients, and several of AChE inhibitors targeting this dysfunction are used for the AD treatment in clinic. However, their therapeutic effects are usually limited to the mild or early stages of AD. Since growing evidences reveal the changes of BChE in both enzymatic activity and expression level in the AD patients, it is proposed that the role of BChE in cholinergic dysfunction may become more pronounced as AD progresses, indicating a great potential for AD treatment using selective BChE inhibitors or dual inhibitors. In the present study, we found that compound exhibited the strongest BChE inhibitory potency and highest selectivity for BChE (IC = 2.34 μM, selective ratio = 55.98), which was much better than that of the reference, galanthamine (IC = 13.18 μM). Compound also presented a significant inhibition (IC = 11.49 μM) and certain selective ratio (>17.41) for BChE. Barbigerone (), which was previously shown to be a major constituent of this species, showed moderate inhibitory activity against BChE (IC = 21.77 μM). In addition, compound was found to be the most potent dual-action inhibitor (IC = 17.14, IC = 5.68 μM). These results suggested that the anti-BChE activity of EtOAc extract of this species might be attributed to these flavonoids ( and ). Unfortunately, the new compound was inactive against both AChE and BChE. In spite of the less beneficial anticholinesterase activity of flavanones, this showed some useful information about the structure-activity relationship. Three rotenone analogs (, and ) presented poor inhibitory activities. The inhibitory activity of compound on BChE was 10-fold higher than that of , indicating that the lack of a methoxy group at the B ring significantly increased the inhibitory effect on BChE but slightly reduced the inhibitory activity against AChE. Compounds and containing methoxy substitution at C-4′ position of the B ring, were found to be less active toward two ChEs when compared with compounds and bearing the hydroxyl group at same position, which revealed the importance of the presence of the hydroxyl group at this position. Owing to the potent performance of BChE inhibition, , , and were selected for further kinetic study to illustrate their BChE inhibitory mechanism on cholinesterases. As shown in , the Lineweaver–Burk plots indicated that the increase of inhibitor concentrations resulted in a decrease in and an increase in , suggesting that three active compounds were all mixed-type BChE inhibitors with inhibition constants () of 3.48, 0.92, and 3.73 μM, respectively. While compound was a competitive inhibitor against AChE ( = 7.39 μM). Their bindings to cholinesterases were further proved by monitoring quenching of the intrinsic protein fluorescence. showed the fluorescence emission spectra of cholinesterases at the presence of , , and at 25 °C, respectively. The BChE peak, with maximum fluorescence intensity of 760.2 a.u., was observed at approximately 337 nm. After compound was added to the BChE solution, the maximum fluorescence intensity significantly decreased with increasing concentrations of , and the same trend was observed between and AChE. The concentrations of for 50% intrinsic fluorescence quenching of AChE and BChE were 29.56 and 2.57 μM, respectively, which were consistent with its IC values for both enzymes. Compounds and also showed strong interactions with BChE, with concentrations of 7.82 and 3.57 μM for 50% intrinsic fluorescence quenching, respectively, further supporting their significant anti-BChE activities.