Inhibitory effect of natural polyphenols on recombinant and endogenous dentinal proteases.

Objectives: This study aimed to investigate the effects of four natural polyphenols on recombinant and endogenous proteases, and to explore their potential mechanisms.

Methods: The interaction of natural polyphenols, namely proanthocyanidins (PA), epigallocatechin-3-gallate (EGCG), quercetin (QUE), and resveratrol (RES) with dentin matrix was observed using SEM. The inhibitory effects of natural polyphenols and the control chlorhexidine (CHX) on matrix metalloproteinases 2 and 8 (MMP-2 and MMP-8) were assessed using fluorometric assay kits. Bacterial collagenase activity was measured by analyzing the loss of dry mass and released hydroxyproline. In situ zymography (ISZ) was conducted to determine matrix-bound protease activity. Furthermore, in silico molecular docking simulations were performed to investigate the interactions between natural polyphenols and proteases.

Results: SEM observation demonstrated the collagen-protective effects of four natural polyphenols against collagenase digestion. However, only PA showed a performance comparable to CHX group on both recombinant MMP-2 and MMP-8. Concerning bacterial collagenase, PA and EGCG exhibited superior inhibitory effects compared to QUE and RES (p < 0.05). The fluorescence intensity of ISZ indicated stronger suppression of endogenous protease activity in the PA and EGCG groups than in the QUE and RES groups. Molecular docking analysis consistently showed that PA and EGCG had higher binding affinities for both MMP2 and MMP8 than QUE and RES.

Conclusion: Natural polyphenols can deactivate both soluble and matrix-bound proteases, the stronger inhibitory effect may be attributed to the formation of more hydrogen bonds.

Clinical Significance: Natural polyphenolic protease inhibitors with more hydroxyphenyl radicals could potentially enhance anti-proteolytic performance and bonding durability.