Investigation on the interaction mechanism of trilobatin with pepsin and trypsin by multi-spectroscopic and molecular docking methods

Pepsin and trypsin are noteworthy for the digestion of proteins in the human body. Trilobatin is a dihydrochalone that has anti-obesity, antioxidant, and anti-diabetes functions. The interaction of trilobatin and pepsin or trypsin is not currently being explored. Therefore, in this research multiple methods involving fluorescence spectroscopy, synchronous fluorescence spectroscopy, ultraviolet–visible (UV–Vis) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) and molecular docking were employed for studying the interaction between trilobatin and pepsin or trypsin. During the interaction between trilobatin and pepsin or trypsin, the quenching type was static quenching and a single binding site existed. The binding constants (K_a) values were 2.177 and 3.028 × 10⁴ L/mol in trilobatin-pepsin and trilobatin-trypsin system at 298 K, respectively. However, the presense of metal ions (Zn²⁺, Ca²⁺ and K⁺) decreased the binding of trilobatin to pepsin and trypsin. The complex between trilobatin and pepsin was generated typically through hydrogen bonding and van der Waals force (ΔH° < 0 and ΔS° < 0), in comparison the complex between trilobatin and trypsin was established chiefly through hydrogen bond and hydrophobic force (ΔH° > 0 and ΔS° > 0). The data from UV–Vis, synchronous fluorescence, FT-IR and CD spectra indicated that trilobatin reduced the hydrophobicity of Tyrosine (Tyr) residues of pepsin and trypsin. Furthermore, trilobatin altered the secondary structures of pepsin and trypsin. Meanwhile, trilobatin changed the conformation of pepsin and trypsin so that the viscosity of the interaction systems decreased with increased of trilobatin concentration. The molecular docking modeling identified that the trilobatin interacted with amino acid residues around pepsin and trypsin. This work consistently showed the interaction between trilobatin and pepsin or trypsin, in addition to offering valuable information for the application of trilobatin.