Bidirectional effects of cuminaldehyde–pepsin interaction on enzymatic and antioxidant activity: Potential implications for gastrointestinal health and disorder modulation

Pepsin is the primary proteolytic enzyme in the stomach and plays a pivotal role in gastric protein digestion, with dysregulation closely associated with gastrointestinal functional disorders. Cuminaldehyde, a dietary phytochemical abundant in Cuminum cyminum (cumin), is widely consumed and recognized for its antioxidant and gastrointestinal bioactivities. In this study, we systematically investigated the interaction between cuminaldehyde and pepsin under physiologically relevant conditions to elucidate its implications for gastric digestion, antioxidant function, and gastrointestinal health. Fluorescence quenching decreased with rising temperature ( K SV 1.04 × 10 5 to 0.19 × 10 5 M −1 ; k q 1.04 to 0.19 × 10 13 M −1 s −1 from 298 to 316 K). Binding constants increased (log K a 4.54–4.98) with binding sites ( n ) 0.87–1.13. Thermodynamic parameters (Δ H° = 42.59 kJ/mol; Δ S° = 229.74 J/mol·K; Δ G° = -25.86 to -30.00 kJ/mol) indicated spontaneous interaction. Secondary structure analysis showed β-sheet reduction (51.23% to 41.44%) and random coil increase (27.67% to 36.68%). Furthermore, kinetic studies showed that activation energy ( E a ) increased (37.33 to 40.23 kJ/mol), while V max decreased (0.2419 to 0.1156 μmol/min/mL), confirming inhibitory effects of cuminaldehyde against pepsin. Importantly, the interaction also modulated the functional properties of cuminaldehyde itself, as its radical-scavenging capacity assessed by DPPH and ABTS assays was attenuated upon binding, likely due to hydrogen-bond interactions involving Gly-109. Molecular dynamics (MD) simulations confirmed the stability of the cuminaldehyde–pepsin complex and revealed localized flexibility changes within the enzyme structure. In conclusion, these findings demonstrate a bidirectional interaction in the gastric environment, whereby cuminaldehyde modulates pepsin-mediated protein digestion while its antioxidant activity is partially compromised through enzyme binding. This study provides mechanistic insight into phytochemical–enzyme interactions during digestion and suggests potential implications for dietary regulation of gastric function and gastrointestinal disorder modulation.