Tri- and Tetrameric Proanthocyanidins with Dentin Bioactivities from Pinus massoniana.

Guided by dentin biomechanical bioactivity, this phytochemical study led to the elucidation of an extended set of structurally demanding proanthocyanidins (PACs). Unambiguous structure determination involved detailed spectroscopic and chemical characterization of four A-type dimers (2 and 4-6), seven trimers (10-16), and six tetramers (17-22). New outcomes confirm the feasibility of determining the absolute configuration of the catechol monomers in oligomeric PACs by one-dimensional (1D) and two-dimensional (2D) NMR. Electronic circular dichroism as well as phloroglucinolysis followed by mass spectrometry and chiral phase high-performance liquid chromatography (HPLC) analysis generated the necessary chiral reference data. In the context of previously reported dentin-bioactive PACs, accurately and precisely assigned 13C NMR resonances enabled absolute stereochemical assignments of PAC monomers via (i) inclusion of the 13C NMR γ-gauche effect and (ii) determination of differential 13C chemical shift values (ΔδC) in comparison with those of the terminal monomer (unit II) in the dimers 2 and 4-6. Among the 13 fully elucidated PACs, eight were identified as new, and one structure (11) was revised based on new knowledge gained regarding the subtle, stereospecific spectroscopic properties of PACs.

Targeting Trimeric and Tetrameric Proanthocyanidins of Cinnamomum verum Bark as Bioactives for Dental Therapies.

The present study elucidated the structures of three A-type tri- and tetrameric proanthocyanidins (PACs) isolated from Cinnamomum verum bark to the level of absolute configuration and determined their dental bioactivity using two therapeutically relevant bioassays. After selecting a PAC oligomer fraction via a biologically diverse bioassay-guided process, in tandem with centrifugal partition chromatography, phytochemical studies led to the isolation of PAC oligomers that represent the main bioactive principles of C. verum: two A-type tetrameric PACs, epicatechin-(2ß→O→7,4ß→8)-epicatechin-(4ß→6)-epicatechin-(2ß→O→7,4ß→8)-catechin (1) and parameritannin A1 (2), together with a trimer, cinnamtannin B1 (3). Structure determination of the underivatized proanthocyanidins utilized a combination of HRESIMS, ECD, 1D/2D NMR, and 1H iterative full spin analysis data and led to NMR-based evidence for the deduction of absolute configuration in constituent catechin and epicatechin monomeric units.

Proanthocyanidin Dimers and Trimers from Vitis vinifera Provide Diverse Structural Motifs for the Evaluation of Dentin Biomodification.

Aimed at exploring the dentin biomodification potential of proanthocyanidins (PACs) for the development of dental biomaterials, this study reports the phytochemical and dental evaluation of nine B-type PACs from grape seed extract (GSE). Out of seven isolated dimers (1-7), four new compounds (2, 3, 5, and 6) involved relatively rare ent-catechin or ent-epicatechin monomeric flavan-3-ol units. Low-temperature NMR analyses conducted along with phloroglucinolysis and electronic circular dichroism enabled unequivocal structural characterization and stereochemical assignment. Additionally, one known (8) and one new (9) B-type trimer were characterized. Differential 13C NMR chemical shifts (Δδ) were used to determine the absolute configuration of 9, relative to the dimers 1 and 2 as the possible constituent subunits. Compared to the dimers, the trimers showed superior dentin biomodification properties. The dimers, 1-7, exhibited pronounced differences in their collagenase inhibitory activity, while enhancing dentin stiffness comparably. This suggests that PAC structural features such as the degree of polymerization, relative and absolute configuration have a differential influence on enhancement of dentin biomechanical and biostability. As mechanical enhancement to dentin and resistance to proteolytic biodegradation are both essential properties functional and stable dentin substrate, the structurally closely related PACs suggest a new metric, the dentin biomodification potential (DBMP) that may rationalize both properties.

Absolute Configuration of Native Oligomeric Proanthocyanidins with Dentin Biomodification Potency.

The structurally complex oligomeric proanthocyanidins (OPACs) are promising biomimetic agents, capable of strengthening the macromolecular backbone of teeth via intermolecular and intermicrofibrillar cross-linking. This study establishes analytical methods capable of determining the absolute configuration of the catechin-type monomeric units of underivatized OPACs. This preserves the capacity of their biological evaluation, aimed at understanding the inevitably stereospecific interactions between the OPACs and dentin collagen. Guided by dental bioassays (modulus of elasticity, long-term stability), two new trimeric and tetrameric A-type OPACs were discovered as dentin biomodifiers from pine (Pinus massoniana) bark: epicatechin-(2ß→O→7,4ß→8)-epicatechin-(2ß→O→7,4ß→8)-catechin (5) and epicatechin-(2ß→O→7,4ß→8)-epicatechin-(2ß→O→7,4ß→6)-epicatechin-(2ß→O→7,4ß→8)-catechin (6), respectively. Combining 1D/2D NMR, HRESIMS, ECD, 1H iterative full spin analysis (HiFSA), and gauge-invariant atomic orbital (GIAO) δ calculations, we demonstrate how 13C NMR chemical shifts (diastereomeric building blocks (A-type dimers)) empower the determination of the absolute configuration of monomeric units in the higher oligomers 5 and 6. Collectively, NMR with ECD reference data elevates the level of structural information achievable for these structurally demanding molecules when degradation analysis is to be avoided. Considering their numerous and deceptively subtle, but 3D impactful, structural variations, this advances the probing of OPAC chemical spaces for species that bind selectively to collagenous and potentially other biologically important biomacromolecules.

Evolution of Quantitative Measures in NMR: Quantum Mechanical qHNMR Advances Chemical Standardization of a Red Clover (Trifolium pratense) Extract.

Chemical standardization, along with morphological and DNA analysis ensures the authenticity and advances the integrity evaluation of botanical preparations. Achievement of a more comprehensive, metabolomic standardization requires simultaneous quantitation of multiple marker compounds. Employing quantitative 1H NMR (qHNMR), this study determined the total isoflavone content (TIfCo; 34.5-36.5% w/w) via multimarker standardization and assessed the stability of a 10-year-old isoflavone-enriched red clover extract (RCE). Eleven markers (nine isoflavones, two flavonols) were targeted simultaneously, and outcomes were compared with LC-based standardization. Two advanced quantitative measures in qHNMR were applied to derive quantities from complex and/or overlapping resonances: a quantum mechanical (QM) method (QM-qHNMR) that employs 1H iterative full spin analysis, and a non-QM method that uses linear peak fitting algorithms (PF-qHNMR). A 10 min UHPLC-UV method provided auxiliary orthogonal quantitation. This is the first systematic evaluation of QM and non-QM deconvolution as qHNMR quantitation measures. It demonstrates that QM-qHNMR can account successfully for the complexity of 1H NMR spectra of individual analytes and how QM-qHNMR can be built for mixtures such as botanical extracts. The contents of the main bioactive markers were in good agreement with earlier HPLC-UV results, demonstrating the chemical stability of the RCE. QM-qHNMR advances chemical standardization by its inherent QM accuracy and the use of universal calibrants, avoiding the impractical need for identical reference materials.

Subtle Chemical Shifts Explain the NMR Fingerprints of Oligomeric Proanthocyanidins with High Dentin Biomodification Potency.

The ability of certain oligomeric proanthocyanidins (OPACs) to enhance the biomechanical properties of dentin involves collagen cross-linking of the 1.3-4.5 nm wide space via protein-polyphenol interactions. A systematic interdisciplinary search for the bioactive principles of pine bark has yielded the trimeric PAC, ent-epicatechin-(4ß→8)-epicatechin-(2ß→O→7,4ß→8)-catechin (3), representing the hitherto most potent single chemical entity capable of enhancing dentin stiffness. Building the case from two congeneric PAC dimers, a detailed structural analysis decoded the stereochemistry, spatial arrangement, and chemical properties of three dentin biomodifiers. Quantum-mechanics-driven (1)H iterative full spin analysis (QM-HiFSA) of NMR spectra distinguished previously unrecognized details such as higher order J coupling and provided valuable information about 3D structure. Detection and quantification of H/D-exchange effects by QM-HiFSA identified C-8 and C-6 as (re)active sites, explain preferences in biosynthetic linkage, and suggest their involvement in dentin cross-linking activity. Mapping of these molecular properties underscored the significance of high δ precision in both (1)H and (13)C NMR spectroscopy. Occurring at low- to subppb levels, these newly characterized chemical shift differences in ppb are small but diagnostic measures of dynamic processes inherent to the OPAC pharmacophores and can help augment our understanding of nanometer-scale intermolecular interactions in biomodified dentin macromolecules.

A dynamic mechanical method to assess bulk viscoelastic behavior of the dentin extracellular matrix.

OBJECTIVES: To develop a protocol for assessment of the bulk viscoelastic behavior of dentin extracellular matrix (ECM), and to assess relationships between induced collagen cross-linking and viscoelasticity of the dentin ECM. METHODS: Dentin ECM was treated with agents to induce exogenous collagen cross-linking: proanthocyanidins (PACs) from Vitis vinifera - VVe, PACs from Pinus massoniana - PMe, glutaraldehyde - (GA), or kept untreated (control). A dynamic mechanical strain sweep method was carried out in a 3-point bending submersion clamp at treatment; after protein destabilization with 4 M urea and after 7-day, 6-month, and 12-month incubation in simulated body fluid. Tan δ, storage (E'), loss (E"), and complex moduli (E*) were calculated and data were statistically analyzed using two-way ANOVA and post-hoc tests (α = 0.05). Chemical analysis of dentin ECM before and after protein destabilization was assessed with ATR-FTIR spectroscopy. RESULTS: Significant interactions between study factors (treatment vs. time points, p < 0.001) were found for all viscoelastic parameters. Despite a significant decrease in all moduli after destabilization, PAC-treated dentin remained statistically higher than control (p < 0.001), indicating permanent mechanical enhancement after biomodification. Covalently crosslinked, GA-treated dentin was unaffected by destabilization (p = 0.873) and showed the lowest damping capacity (tan δ) at all time points (p < 0.001). After 12 months, the damping capacity of PMe and VVe groups decreased significantly. Changes in all amide IR resonances revealed a partial chemical reversal of PAC-mediated biomodification. SIGNIFICANCE: Viscoelastic measurements and IR spectroscopy aid in elucidating the role of inter-molecular collagen cross-linking in the mechanical behavior of dentin ECM.

Evidence to the role of interflavan linkages and galloylation of proanthocyanidins at sustaining long-term dentin biomodification.

OBJECTIVES: The interactivity of proanthocyanidins (PACs) with collagen modulates dentin matrix biomechanics and biostability. Herein, PAC extracts selected based on structural diversity were investigated to determine key PAC features driving sustained effects on dentin matrices over a period of 18months. METHODS: The chemical profiles of PAC-rich plant sources, Pinus massoniana (PM), Cinnamomum verum (CV) and Hamamelis virginiana (HV) barks, as well as Vitis vinifera (VV) seeds, were obtained by diol HPLC analysis after partitioning of the extracts between methyl acetate and water. Dentin matrices (n=15) were prepared from human molars to determine the apparent modulus of elasticity over 18months of aging. Susceptibility of the dentin matrix to degradation by endogenous and exogenous proteases was determined by presence of solubilized collagen in supernatant, and resistance to degradation by bacterial collagenase, respectively. Data were analyzed using ANOVA and Games-Howell post hoc tests (α=0.05). RESULTS: After 18months, dentin matrices modified by PM and CV extracts, containing only non-galloylated PACs, were highly stable mechanically (p<0.05). Dentin matrices treated with CV exhibited the lowest degradation by bacterial collagenase after 1h and 18months of aging (p<0.05), while dentin matrices treated with PM showed the least mass loss and collagen solubilization by endogenous enzymes over time (p<0.05). SIGNIFICANCE: Resistance against long-term degradation was observed for all experimental groups; however, the most potent and long-lasting dentin biomodification resulted from non-galloylated PACs.

Centrifugal partition chromatography enables selective enrichment of trimeric and tetrameric proanthocyanidins for biomaterial development.

Proanthocyanidins (PACs) find wide applications for human use including food, cosmetics, dietary supplements, and pharmaceuticals. The chemical complexity associated with PACs has triggered the development of various chromatographic techniques, with countercurrent separation (CCS) gaining in popularity. This study applied the recently developed DESIGNER (Depletion and Enrichment of Select Ingredients Generating Normalized Extract Resources) approach for the selective enrichment of trimeric and tetrameric PACs using centrifugal partition chromatography (CPC). This CPC method aims at developing PAC based biomaterials, particularly for their application in restoring and repairing dental hard tissue. A general separation scheme beginning with the depletion of polymeric PACs, followed by the removal of monomeric flavan-3-ols and a final enrichment step produced PAC trimer and tetramer enriched fractions. A successful application of this separation scheme is demonstrated for four polyphenol rich plant sources: grape seeds, pine bark, cinnamon bark, and cocoa seeds. Minor modifications to the generic DESIGNER CCS method were sufficient to accommodate the varying chemical complexities of the individual source materials. The step-wise enrichment of PAC trimers and tetramers was monitored using normal phase TLC and Diol-HPLC-UV analyses. CPC proved to be a reliable tool for the selective enrichment of medium size oligomeric PACs (OPACs). This method plays a key role in the development of dental biomaterials considering its reliability and reproducibility, as well as its scale-up capabilities for possible larger-scale manufacturing.

A galloylated dimeric proanthocyanidin from grape seed exhibits dentin biomodification potential.

Grape seeds are a rich source of polyphenols, especially proanthocyanidins (PACs), and are also known for the presence of galloylated oligomeric PACs (OPACs). The present study focuses on the phytochemical methodology for grape seed (O)PACs and their potential role as dentin biomodifiers to be used in restorative and reparative dentistry. A new method using centrifugal partition chromatography (CPC) was developed for the preparative separation of the grape seed (O)PACs. Orthogonal phytochemical profiling of the resulting CPC fractions was performed using C18 and diol HPLC, normal phase HPTLC, and IT-TOF MS analysis. A galloylated procyanidin dimer (1) was isolated from a CPC fraction in order to evaluate its potential to enhance dentin bio-mechanical properties. Moreover, it helped to evaluate the impact of the galloyl moiety on the observed bioactivity. Structure elucidation was performed using ESI-MS, 1D and 2D NMR analyses. For the first time, (1)H iterative full spin analysis (HiFSA) was performed on this type of molecule, enabling a detailed proton chemical shift and coupling constant assignment. The CPC fractions as well as 1 showed promising results in the dentin stiffness bioassay and indicate that they may be used as dental intervention biomaterial.

Dentin biomodification: strategies, renewable resources and clinical applications.

OBJECTIVES: The biomodification of dentin is a biomimetic approach, mediated by bioactive agents, to enhance and reinforce the dentin by locally altering the biochemistry and biomechanical properties. This review provides an overview of key dentin matrix components, targeting effects of biomodification strategies, the chemistry of renewable natural sources, and current research on their potential clinical applications. METHODS: The PubMed database and collected literature were used as a resource for peer-reviewed articles to highlight the topics of dentin hierarchical structure, biomodification agents, and laboratorial investigations of their clinical applications. In addition, new data is presented on laboratorial methods for the standardization of proanthocyanidin-rich preparations as a renewable source of plant-derived biomodification agents. RESULTS: Biomodification agents can be categorized as physical methods and chemical agents. Synthetic and naturally occurring chemical strategies present distinctive mechanism of interaction with the tissue. Initially thought to be driven only by inter- or intra-molecular collagen induced non-enzymatic cross-linking, multiple interactions with other dentin components are fundamental for the long-term biomechanics and biostability of the tissue. Oligomeric proanthocyanidins show promising bioactivity, and their chemical complexity requires systematic evaluation of the active compounds to produce a fully standardized intervention material from renewable resource, prior to their detailed clinical evaluation. SIGNIFICANCE: Understanding the hierarchical structure of dentin and the targeting effect of the bioactive compounds will establish their use in both dentin-biomaterials interface and caries management.