Mechanism of anthocyanins from Aronia melanocarpa in regulating pancreatic amylase activity and improving glucose tolerance in vivo: A combined experimental and molecular dynamics simulation study.

With the escalating global incidence of diabetes posing a significant public health risk, pancreatic amylase (a pivotal regulator of starch digestion and postprandial glucose metabolism) has emerged as a key target for inhibitor development. Although anthocyanins from Aronia melanocarpa exhibit promising inhibitory potential, their precise interaction mechanisms with pancreatic amylase, structure-activity relationships, and digestive stability remain unclear. This study systematically integrated virtual screening, 100 ns molecular dynamics (MD) simulations, and comprehensive in vitro/in vivo validation to elucidate the regulatory mechanism. Computational and experimental analyses identified cyanidin-3-glucoside (C3G) as the most potent monomer among four characterized anthocyanins (C3G, C3A, C3Ga, C3X), with an IC₅₀ of (38.5 ± 3.2) μM. Mechanistically, C3G exerts competitive inhibition by reversibly binding to the enzyme's active site (ASP356, ASP197), and MD simulations confirmed the stability of the C3G-amylase complex (RMSD <2.0 Å). Fluorescence spectroscopy further verified that C3G binding induces conformational changes in the enzyme. Simulated digestion experiments demonstrated that C3G retained 67 % of its inhibitory activity post oral-intestinal digestion, exhibiting significantly higher stability than C3A (54.84 ± 1.89 % retention). In vivo studies on high-sugar/high-fat diet-induced mice showed that A. melanocarpa anthocyanins improved oral starch tolerance and regulated blood glucose with progressive efficacy as dosage increased, without affecting body weight. These findings clarify the molecular basis of A. melanocarpa anthocyanins in pancreatic amylase regulation and validate C3G as a stable, effective functional ingredient， providing robust empirical support for its potential in blood glucose-targeted functional foods.