Fig 1.
Molecular structures of catechins and structurally related polyphenolic compounds. (-)-epicatechin (a), (-)-epigallocatechin (b), (-)-epicatechin-3-gallate (c), (-)-epigallocatechin-3-gallate (d), methyl gallate (e), and quercetin-3-O-rutinoside (f).
Fig 2.
Analysis of (-)-epigallocatechin gallate (2.5 mmol/L) upon titration with salivary mucin.
(a) Excerpts of the HPLC-UV chromatograms (λ = 276 nm) showing equal peak areas for EGCG that are independent of the protein concentration. (b) 1H NMR (NOESYPRESAT, H2O/Buffer (9:1, v/v), 300 K) spectra of EGCG in phosphate buffer (pH 7.0). The NMR signals of free EGCG in the absence of mucin are shown in spectrum b1. Sharp proton signals are clearly visible and are assigned to the molecular structure of EGCG being well in line with literature data [32]. As mucin is titrated to the sample, the proton signals of EGCG decrease (spectra b2−b10) until they merge with the baseline. NMR signals between 4 and 5.5 ppm are partially suppressed by the water suppression technique.
Fig 3.
Fitted binding isotherms of polyphenols titrated with increasing levels of salivary mucin.
The mole fraction of polyphenol bound to mucin is plotted against the protein concentration. The concentration of EC (a), EGC (b), ECG (c), EGCG (d), methyl gallate (e), and rutin (f) was kept constant (2.5 mmol/L), whereas salivary mucin was titrated from 0 to 2.5 μmol/L or until the bound mole fraction of the ligand reached a plateau. The approximate mucin content of human whole saliva is marked by an arrow. The intercept of the fitted binding isotherm with the horizontal gray line marks the half-maximum binding concentration of the protein (BC50). Error bars denote the standard deviation obtained from integrating all the distinct, non-overlaid proton signals of each compound (see methods for a list of consulted proton resonance signals). For methyl gallate only one discrete 1H resonance signal is available for integration. *Error bars in (e) denote the technical error of two independent measurements. Data points behind means are provided in S1 Table. Binding isotherms, the BC50 value, and the shape parameter α were fitted with the Gnuplot software (Version 5.0).
Table 1.
Fit parameters of polyphenol–mucin binding isotherms.
Fig 4.
Mole fractions of polyphenols bound to human saliva proteins as assessed by single comparative qHNMR measurements.
Aliquots (10 μL, 2.5 mmol/L) of the polyphenolic compounds EC, EGC, ECG, EGCG, methyl gallate, rutin, and saccharin were each mixed with pooled and centrifuged, non-stimulated saliva (990 μL). As crude saliva was previously blended with phosphate buffer in a 9:1 ratio (v/v), the total saliva content was 89.1%. Samples lacking saliva served as references. Error bars denote the standard deviation obtained from two independent measurements of biological replicates, integrating all distinct, non-overlaid proton signals of each ligand (see methods for a list of consulted proton resonance signals and S2 Table for the data points behind means). Saccharin showed no binding (n.b.) with human saliva.
Table 2.
Pairwise comparison matrix to assess the relative astringency of polyphenols.
Fig 5.
Unbound (-)-epigallocatechin gallate (EGCG) in pure buffer (reference), 10% (v/v) human whole saliva, 1% (w/v) carboxymethyl cellulose (CMC), and a combination of both.
Unbound EGCG was quantified by single qHNMR measurements and is expressed as mole fractions. Error bars denote the standard deviation as obtained from integrating all the distinct, non-overlaid proton signals of EGCG. Data points behind means are provided in S3 Table.