In light of this, we did the present work aiming to emphasize GSE constituents’; collagen-stabilizing activity in clinically relevant settings. The experimental design features ultra-thin (6-μm) dentin films that mimic the acid-etched dentin layer in a total-etch procedure, and short treatment time (1 min) that is clinically feasible. In addition, using thin specimens facilitates the removal of treatment reagent that is physically trapped in the spongy demineralized dentin collagen matrix rather than chemically bound to it. It is believed that the effect of physically-trapped compounds on dentin bonding diminishes over time due to oral fluid exchange, and is therefore of little interest to us as our ultimate goal is to improve long-term bonding to dentin in clinical situations. Overall, six monomeric species, two dimeric species (Fig. 1), a low molecular weight fraction (PALM) of commercially available GSE, as well as the original GSE and a high molecular weight fraction (PAHM) were examined representing a gradually increased average molecular size of treatment reagents. The tested null LY2228820
is that the structure of the tested chemicals has no effect on their collagen-stabilizing capability.
Fig. 1. Structure of monomeric and dimeric PA-related species investigated.Figure optionsDownload full-size imageDownload high-quality image (181 K)Download as PowerPoint slide
2. Materials and methods
Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), including the 6 monomeric species (+)-catechin (pCT), (?)-catechin (CT), (?)-epicatechin (EC), (?)-epigallocatechin (EGC), (?)-epicatechin gallate (ECG) and (?)-epigallocatechin gallate (EGCG) (Fig. 1). The representative EC-EC dimer, procyanidin B2 was purchased from Chromadex (Irvine, CA, USA). The representative pCT-pCT dimer (Fig. 1) was synthesized from pCT following a published procedure . MegaNatural? Gold grape seed
extract (Lot #: 05592502-01) was donated by the manufacturer (Polyphenolics, Madera, CA, USA). All reagents were used as received.
2.2. Preparation and characterization of PALM and PAHM
GSE was separated into a low molecular weight fraction (PALM) and high molecular weight fraction (PAHM) using adapted methods of extraction  and preparative size exclusion
chromatography (SEC) . In a typical procedure, 2.3 g of grape seed extract was dissolved in 200 mL of deionized water, which was subsequently extracted three times with ethyl acetate. The organic phase was lyophilized to remove the solvent, and approximately 0.27 g of residue was collected as PALM. The water phase was air-dried, re-dissolved in 20 mL of methanol, and loaded on a column (250 × 16 mm internal diameter) packed with Toyopearl TSK HW-40F resin (Tosoh, Japan). The column was sequentially eluted by methanol, water and acetone/water mixtures (20/80, 30/70, 40/60, 60/40, v/v). The resultant fractions were lyophilized and the last fraction (approximately 0.7 g) was designated as PAHM.