Antiglycation of Insulin by Selected Six Bioactive Peanut Sprout Stilbenoids and Demonstration of Methylglyoxal-Mediated Biphasic Insulin Glycation by O-Phenylenediamine Derivatization.

Protecting insulin molecules from glycation by the dietary ingredients is critical to alleviate diabetes-associated metabolic complications. Six peanut sprout stilbenoids were selected for antiglycation determination with bovine insulin mediated by methylglyoxal (MG), glucose, and fructose. Monitoring at 215 nm via HPLC analysis revealed that all test compounds exhibited inhibitory activities with structure-activity dependency, among those arachidin-3 performing the most potent efficacy (p < 0.05). Notably, a biphasic dose-response was observed, indicating that MG-mediated insulin modification increased as MG concentrations decreased from 500 to 5 mM and then declined as MG concentrations dropped to 0.05 mM. This was supported by insulin band-intensity changes shown in Tricine SDS-PAGE pattern. O-phenylenediamine (OPD) was utilized as a trapping probe to form MG-OPD adducts for characterization and isolation. In high-MG concentrations, a novel intermediate: 2-(hydroxymethyl)-1,2,3,4-tetrahydroquinoxaline-2,3-diol (2HMQL) was isolated, identified and merited to speculate its precursor compounds existing in the MG equilibrium complex with less reactive hydrates or tautomers (e.g., dihydroxyacetone and/or hydroxypyruvaldehyde) which protect insulin integrity from glycation. It is of merit to demonstrate that identification of 2HMQL provides a proposed equilibrium mechanism to elucidate the biphasic MG-mediated insulin glycation.