SAR of grape seed galloylated proanthocyanidins versus dentin stiffening activity

To extend the life of a dental restoration, a strong and stable dentin-collagen matrix is a requisite. The strength of dentin can be evaluated with a dentin stiffness assay (DSA), which we perform on a Shimadzu EZ Graph, testing the mechanical strength of a demineralized dentin beam after treating for 1 hr with samples from different sources. In our screen of natural sources, the enriched proanthocyanidins (PACs) of grape seed extract (GSE) showed an 11-fold increase from the blank in the DSA. After several fractionations, the activity of subfractions showed a 14-fold increase in DSA compared with the blank. In our previous study, the monogalloylated PAC monomers from green tea (GT) exhibited higher activity than nongalloylated PAC monomers in the DSA. Literature reported that GSE contained both A- and B-type galloylated PACs. In order to detect whether galloylated PACs existed in our subfractions, a negative ESI LCMS method was developed on the basis of a neutral loss of a galloyl fragment to scan precursors. After verification of the method with the NIST certificate GT extract, it was used to analyze PAC-enriched GSE subfractions. The results showed several peaks in these active fractions. On the basis of LC retention time and mass-to-charge ratio, these hits were tentatively identified as a monogalloylated PAC monomer ECG, a B-type dimer and digalloylated PAC A- and B-type dimers. An internal reference standard, synthesized in-house, was used to reduce system errors caused by different ionization responses, and to generate comparable data. These data were used to establish the relationship between galloylated PAC oligomers and dentin stiffness activity.