Inhibitory effects of carvacrol on glucansucrase from Streptococcus mutans and salivary α-amylase: in silico and in vitro studies

Abstract

Background/aim: Streptococcus mutans produces glucansucrase, an enzyme that converts sucrose into lactic acid, which lowers the pH in the oral environment and leads to tooth enamel demineralization, a key factor in dental caries. Additionally, glucansucrase facilitates the formation of extracellular polysaccharides, which promote bacterial adhesion to tooth surfaces. This study investigates the inhibitory effects of carvacrol, a natural compound, on glucansucrase activity both in vitro and in silico.

Materials and methods: Glucansucrase enzyme was purified from S. mutans. The inhibitory effects of carvacrol against glucansucrase enzyme were investigated both in vitro and in silico.

Results: In the presence of 50 mM carvacrol, glucansucrase and salivary amylase activities were reduced by 51.25% and 14.85%, respectively. Carvacrol did not significantly inhibit (4.73%) the salivary amylase enzyme at 10 mM. Glucansucrase activity decreased by 51.63% in the presence of 10 mM acarbose, which was used as a positive control in glucansucrase enzyme studies. Acarbose inhibited salivary amylase with 82.54% loss of enzyme activity in the presence of 1 mM acarbose. The docking score obtained for carvacrol was -5.262 kcal/mol, while that obtained for acarbose was -6.084 kcal/mol. We carried out molecular dynamics simulation studies for 100 ns to determine the stability of carvacrol in the active site of the protein. Carvacrol demonstrated stable binding to glucansucrase with hydrogen bonds and interactions at key residues (ASP477, GLN960, and ASP909), confirmed by molecular dynamics simulations. Carvacrol remained stable between 16 and 100 ns.

Conclusion: Carvacrol selectively inhibits glucansucrase without significantly affecting salivary amylase, making it a more targeted inhibitor compared to acarbose, which inhibits both enzymes. Docking studies indicated that while carvacrol has a lower binding affinity than acarbose, its stable interaction with the enzyme suggests sustained inhibitory action. These findings highlight carvacrol as a promising natural compound for preventing dental caries, offering a more selective alternative to traditional inhibitors. Further in vivo studies are necessary to assess its therapeutic efficacy and safety in clinical applications for oral health.

Keywords: Carvacrol; Streptococcus mutans; amylase; biofilm; glucansucrase.

Figure 1

Structures of acarbose (A) and carvacrol (B) (obtained from https://go.drugbank.com/).

Figure 2

Bacterial growth and enzyme production versus time.

Figure 3

Inhibitory effects of carvacrol and acarbose on purified glucansucrase.

Figure 4

Inhibitory effects of carvacrol and acarbose on salivary amylase.

Figure 5

The 2D binding modes of carvacrol and acarbose in the active site.

Figure 6

Molecular dynamics simulation process for carvacrol 3AIC. Blue: Cα (RMSD evolution of the protein). Pink: Lig Fit Prot (RMSD of the ligand in the case of the protein–ligand complex).

Figure 7

(A) Fraction of residue interactions obtained during molecular dynamics stimulation with carvacrol in 3AIC. (B) Interactions of carvacrol with the residues in each trajectory frame in 3AIC.