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

Abstract

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-(2β→O→7,4β→8)-epicatechin-(4β→6)-epicatechin-(2β→O→7,4β→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.

Figure 1.

Structures of the oligomeric proanthocyanidins (OPACs), 1-3, isolated from C. verum bark, in comparison with their core A-type dimer, proanthocyanidin A2. In addition to the classical chemical formulas, the graphical PAC Block ARray representations (PACBAR; bottom) along with the text-based macro and micro PACBAR codes.

Figure 2.

Determination of the absolute configuration of 1 via comparison of ¹³C chemical shift differences with those of its four stereochemically possible A-type dimeric PAC moieties.

Figure 3.

The congruence of the observed (red, methanol-d₄, 900 MHz) and calculated (blue) ¹H NMR spectra of OPACs 1 (A) and 2 (B) demonstrates the validity of the HiFSA profiles of the two isolates.

Figure 4.

The ROESY spectrum (900 MHz) shows exchange correlation cross peaks between the two atropisomers of 3 giving rise to signals that exhibit the same phase as the diagonal (red; “in-phase” cross peaks). The major and minor atropisomers are denoted as M and m, respectively.

Figure 5.

Stacked ¹H NMR spectra of 3 acquired at various temperatures in methanol-d₄ (800 MHz).

Figure 6.

Quantitative and qualitative ¹H NMR HiFSA-based determination of the major (A) and minor rotamers (B) of 3. The experimental (observed, red) spectrum was acquired at 900 MHz in methanol-d₄.

Figure 7.

Biomodification potential by two relevant bioassays: (A) fold increase observed in the apparent modulus of elasticity (E) of the dentin matrix; (B) biodegradation rates (in %) of dentin specimens treated with 1–3. The Ω symbol depicts statistically significant differences between groups treated with 1-3 and control for both E (p ≤ 0.001) and biodegradation (p < 0.001).

Figure 8.

The ¹H NMR spectra of a 70% acetone extract, a trimer and tetramer enriched fraction (3+4 cut), and the isolates 1-3 from C. verum. The fraction 3+4 cut (red) was prepared in a prior study and its ¹H NMR spectrum is shown for comparison. The ¹H NMR spectra of the 70% acetone extract and the 3+4 cut were acquired under the same conditions (concentration, volume, NMR acquisition parameters, 600 MHz, methanol-d₄). The ¹H NMR spectra of 1-3 were acquired at 900 MHz in methanol-d₄.