Galloyl moieties enhance the dentin biomodification potential of plant-derived catechins

Abstract

Proanthocyanidin-rich plant-derived agents have been shown to enhance dentin biomechanical properties and resistance to collagenase degradation. This study systematically investigated the interaction of chemically well-defined monomeric catechins with dentin extracellular matrix components by evaluating dentin mechanical properties as well as activities of matrix metalloproteinases (MMPs) and cysteine-cathepsins (CTs). Demineralized dentin beams (n=15) were incubated for 1h with 0.65% (+)-catechin (C), (-)-catechin gallate (CG), (-)-gallocatechin gallate (GCG), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG). The modulus of elasticity (E) and the fold increase in E were determined by comparing specimens at baseline and after treatment. Biodegradation rates were assessed by differences in percentage of dry mass before and after incubation with bacterial collagenase. The inhibition of MMP-9 and CT-B by 0.65, 0.065 and 0.0065% of each catechin was determined using fluorimetric proteolytic assay kits. All monomeric catechins led to a significant increase in E. EGCG showed the highest fold increase in E, followed by ECG, CG and GCG. EGCG, ECG, GCG and CG significantly lowered biodegradation rates and inhibited both MMP-9 and CT-B at a concentration of 0.65%. Overall, the 3-O-galloylated monomeric catechins are clearly more potent than their non-galloylated analogues in improving dentin mechanical properties, stabilizing collagen against proteolytic degradation, and inhibiting the activity of MMPs and CTs. The results indicate that galloylation is a key pharmacophore in the monomeric and likely also in the oligomeric proanthocyanidins that exhibit high cross-linking potential for dentin extracellular matrix.

Keywords: Collagen; Cross-linking; Cysteine-cathepsins; MMP; Proanthocyanidins.

Figure 1

Chemical structures of the monomeric catechins. The two aromatic rings of the core are assigned as A- and B-ring, while the C-ring is a dihydropyrane heterocycle. The circles show the gallo substitution patterns of the B-ring (full circle) and the galloyl moieties attached to C-3-OH (dashed circle).

Figure 2

Pearson correlation coefficient (r) comparing fold increase and biodegradation rates (%) observed for all monomeric catechins.

Figure 3

Inhibition of proteases (%) after incubation with different concentrations of each catechin (after 4 hrs of incubation). A: inhibition of MMP-9 by 0.65% catechins; B: inhibition of MMP-9 by 0.065% catechins; C: inhibition of MMP-9 by 0.0065% catechins; D: inhibition of CT-B by 0.65% catechins; E: inhibition of CT-B by 0.065% catechins; F: inhibition of CT-B by 0.0065% catechins. ( − ): negative control group (inhibitor), C: (+)-catechin, CG: (−)-catechin gallate, GCG: (−)-gallocatechin gallate, EC: (−)-epicatechin, ECG: (−)-epicatechin gallate, EGC: (−)-epigallocatechin, and EGCG: (−)-epigallocatechin-3-gallate. Bars with symbols show statistical significant differences when compared to positive control groups (MMP-9 and CT-B) (p < 0.05). Different symbols indicate statistical significant differences between catechins (p < 0.05).

Figure 4

Comparison of MM2 force field minimized three-dimensional structures of the galloylated catechin, (−)-gallocatechin gallate (GCG), and its C-3 epimer, (−)-epigallocatechin-3-gallate (EGCG), shows the impact of the epimerism on the overall molecular shape. While GCG conformers are potentially stabilized via intramolecular π-stacking, the B- and D-rings in EGCG are anti-parallel.