Activity-oriented isolation of nine α‑glucosidase inhibitors from the Rheum tanguticum waste and interaction mechanism analysis
Weihang Lu, Tao Chen, Cheng Shen, Hailun Feng, Changdong Lin, Zhibo Song, Shuo Wang, Juyuan Luo, Yulin Li
Abstract
The recycling of waste from perennial medicinal crops can enhance economic benefits and promote sustainable development. Aim to develop efficient strategies for isolating α-glucosidase inhibitors from R. tanguticum waste and elucidate their molecular interactions mechanism. An off-line two-dimensional high-speed counter-current chromatography (HSCCC), combined with a multi-separation mode and assisted by liquid chromatograph coupled with mass spectrometer for solvent system optimization, was employed for the rapid and efficient isolation of bioactive compounds. Preparative high performance liquid chromatography (HPLC) was subsequently employed to purify partially isolated fractions. The molecular interaction mechanisms were investigated through various spectroscopic techniques, molecular docking, and molecular dynamics simulations. A novel methylene-bridged flavonoid dimer (rheumbisflavonoid A), along with six flavonoids and a binary mixture, were successfully isolated using first-dimensional HSCCC coupled with a dual-mode elution and preparative HPLC. The binary isomeric mixture was efficiently separated in the second-dimensional HSCCC using a multiple-injection mode. Rheumbisflavonoid A demonstrated stronger α-glucosidase inhibitory activity by binding to the enzyme's active site, forming a stable complex through van der Waals interactions and hydrogen bonding. Furthermore, it altered the microenvironment of critical amino acid residues (tyrosine, tryptophan) and the peptide backbone, resulting in protein’s fluorescence quenching. Notably, the compound significantly decreased the α-helix content of the protein, leading to enzyme inactivation. This study presents a promising approach for developing hypoglycemic products from the waste resources of R. tanguticum . • One novel rare methylene bridging bisflavonoid with the best A-GIA was found. • Interaction mechanism was explained by spectroscopies, molecular docking and MDs. • LC-MS was used to recognition and quantitation of HPLC co-efflux compounds. • Off-line 2D LLC coupled multi-mode was established to isolate nine flavonoids. • The study offers technology for development of AG inhibitors from Rhubarb waste.