Objectives . To isolate the active compound on a proanthocyanidin-rich (PA) extract used to improve the mechanical properties of dentin.
Materials and methods . The polymeric PAs were fractionated from grape seed extract on Sephadex LH-20 column (0.75 ID × 12 in.) using different solvents systems (3 gradients of methanol–water, 100% methanol and 70% acetone–water). The first fractionation provided 35 fractions (Fr), which were combined into 5 Fr, based on the results of TLC (Thin Layer Chromatography) analysis. Second fractionation followed the same protocol and the thirty-five fractions were combined into twenty. One hundred and fifty fragments (1.7 mm × 0.5 mm) of sound coronal dentin were fully demineralized and treated with the respective water dissolved fraction. Specimens were immersed in their respective solution and tested at baseline, 10 min, 30 min and 60 min. The data were collected and statistically analyzed by two-way ANOVA and Tukey’s tests at 95% confidence level. Three-point bend stiffness measurments of demineralized dentin matrix was used as a bioassay to detect the active compounds.
Results . ANOVA from the first fractionation showed statistically significant interaction for the factors (time and fractions, p < 0.001) and differences within each factor ( p < 0.001). Baseline values varied from 7.16 (3.17) to 8.50 (4.71) MPa; 60 min stiffness values were: Fr 1 = 12.71 (5.63), Fr 2 = 10.27 (3.94), Fr 3 = 10.28 (4.57), Fr 4 = 63.67 (40.57) and Fr 5 = 48.09 (28.29). The second fractionation was executed to investigate fractions 4 and 5 which presented the highest stiffness values and therefore more active compounds. The results of the second fractionation (total 20 fractions) were significantly affected by the type of fraction ( p < 0.01) and exposure time ( p < 0.01). The interaction between factors was not statistically significant ( p = 0.279). Baseline values and standard deviations (MPa) ranged from 4.13 (1.43) to 9.25 (5.02); 10 min from 4.31 (1.52) to 18.88 (3.32); 30 min from 3.55 (0.3) to 29.75 (18.19); 60 min from 4.08 (0.49) to 34.57 (20.95). Nine fractions of the second fractionation increased the elastic modulus of dentin collagen after 1 h treatment ( p < 0.05). Data indicates that there may be more than one active compound.
Conclusions . PA extracts consist of a complex mixture of oligomers and polymers composed of the monomeric flavan-3-ols catechin, epicatechin and their gallates. The characterization of these compounds could bring information on their role as collagen cross-linkers. Only specific fractions could interact with dentin collagen, showing that the PA degree of polymerization influences the PA–collagen interaction observed by changes on the mechanical properties. High molecular weight oligomers appear to be the active compound for PA-dentin matrix interaction.
Research funded by CAPES (1880/08-0) NIH-NIDCR (#DE017740).