Inhibitory mechanism of O-methylated quercetins, highly potent -secretase inhibitors isolated from Caragana balchaschensis (Kom.) Pojark

Ethnopharmacological relevance: Caragana has a standing history of implementation in Traditional Chinese Medicine (TCM). Most species of this genus have been explored for multi-functional purposes, such as promoting blood circulation and curing neuralgia, fatigue, migraine, arthritis, and vascular hypertension (Meng et al., 2009). Among them, the well-known species C. sinica showed the most promising potential to increase the expression of ADAM10 among 313 tested medicinal plants, which is one of the promising approach for the treatment of Alzheimer's disease (AD). (Schuck et al., 2015). Aim of this study: The aim of this work is to explore beta-secretase inhibitory activity of compounds isolated from the aerial part of endemic Caragana balchaschensis (Kom.) Pojark. We provided a full characterization of their inhibitory mechanisms, binding affinities, and binding modes. Materials and methods: The isolation of quercetin derivatives was accomplished by various chmmatographical approaches and their structures were annotated by spectroscopic analysis. The detailed kinetic behavior of beta-secretase inhibitors was determined by estimation of kinetic parameters (K-m, V-max, K-I, and K-IS). Binding affinities (K-SV) and binding modes of inhibitors were elucidated by fluorescence quenching and molecular docking studies, respectively. Results: O-methylated quercetins (2-7) were significantly effective in beta-secretase inhibition with IC50 ranging from 1.2 to 6.5 mu M. The most active one (6) was 20-fold effective than the mother skeleton, quercetin. The O-methyl motif was a critical factor in beta-secretase inhibition: tri-O-methylated (1.2 mu M) > di-O-methylated (3.5 mu M) > mono-O-methylated (6.5 mu M) > quercetin (25.2 mu M). In the kinetic study, all quercetins (1-7) showed a noncompetitive inhibition, but glucoside ones (8 and 9) were mixed type I inhibitors. The binding affinities (K-SV) were agreed with inhibitory potencies. The O-methylated quercetins were annotated as the most natural abundant metabolites in the aerial part by LC-ESI-TOF/MS. Binding modes of inhibitors to enzyme were elucidated by molecular docking experiments. Conclusion: This study disclosed that most of the major phenolic metabolites of the aerial part of C. balchaschensis are O-methylated quercetins, which have a significant inhibitory effect on beta-secretase, which is a critical factor for AD.