Exploring the binding effects and inhibiting mechanism of hyperoside to lipase using multi-spectroscopic approaches, isothermal titration calorimetry, inhibition kinetics and molecular dynamics

Abstract

Hyperoside (HYP) is a flavonoid with various physiological activities. The present study examined the interaction mechanism between HYP and lipase using multi-spectrum and computer-aided techniques. Results demonstrated that the force type of HYP on lipase was mainly hydrogen bond, hydrophobic interaction force, and van der Waals force, and HYP had an excellent binding affinity with lipase at 1.576 × 10⁵ M^-1. HYP dose-dependently inhibited lipase in the inhibition experiment, and its IC₅₀ value was 1.92 × 10^-3 M. Moreover, the results suggested that HYP could inhibit the activity by binding to essential groups. Conformational studies indicated that the conformation and microenvironment of lipase were slightly changed after the addition of HYP. Computational simulations further confirmed the structural relationships of HYP to lipase. The interaction between HYP and lipase can provide ideas for the development of functional foods related to weight loss. The results of this study help comprehend the pathological significance of HYP in biological systems, as well as its mechanism.

Fig. 1. (A) The fluorescence spectra of lipase in different concentrations of HYP (a–h: 0–4.2 × 10⁻⁵ M) at 298 K. The insert diagram displays the corresponding double-logarithmic regression plot. (B) The original data on the sequential titration of HYP solutions into lipase solution. (C) Time-resolved fluorescence decay curves of free lipase and the HYP–lipase complex. (D) The integrated heat results of the titration after correction for heat dilution against the molar ratio, and the solid line 1represents the best fitting curve.

Fig. 2. (A, B) The synchronous fluorescence spectra of HYP at varying concentrations (0–4.2 × 10⁻⁵ M) and lipase were obtained at Δλ = 15 and 60 nm. (C, D) The 3D fluorescence spectra of free lipase and the HYP–lipase complex.

Fig. 3. (A) The inhibition of lipase by HYP with increasing HYP concentration. (B) The relationship between the substrate and V₀ at different HYP concentrations. (C) The FT-IR spectra of free lipase and the HYP–lipase complex, C_lipase = 2.0 × 10⁻⁴ M. (D) The CD of lipase with and without different HYP concentrations 3, C_lipase = 2.0 × 10⁻⁴ M.

Fig. 4. The 3D depiction of the HYP binding modalities with lipase. (B) Two–dimensional representation of the interaction maps between HYP and lipase.

Fig. 5. (A) RMSD value of the MD simulation of the free lipase, HYP–lipase complex and free HYP systems. (B) R_g value of the MD simulation of the free lipase, and HYP–lipase complex systems. (C, D) Changes in the secondary structure content of free lipase and HYP–lipase complex within 100 000 ps. (E) RMSFs of the MD simulations of the free lipase and HYP–lipase complex systems.

Fig. 6. (A) ¹H NMR spectrum of HYP alone (1.0 × 10⁻³ M). (B, C) The off-resonance spectra and STD-NMR of the HYP–lipase system (C_lipase = 2.0 × 10⁻⁵ M, and C_HYP = 1.0 × 10⁻³ M).