Fig 1.
Examples of condensed tannins and a galloylated flavan-3-ol.
(A) a B-type condensed tannin; (B) an A-type condensed tannin, epicatechin(4ß→8, 2ß→O→7)-epicatechin; and (C) a flavan-3-ol monomer, epicatechin gallate.
Table 1.
Condensed tannin (CT) contents, mean degrees of polymerisation (mDP), average molecular weights (amw), procyanidin/prodelphinidin (PC/PD) and cis-/trans-flavan-3-ol ratios, molar percentages of galloylation and A-type linkages in fractions isolated from various plant materials.
Note: Results from a few of these fractions were reported previously [4–9, 39] and are included here for clarity purposes.
Fig 2.
The effect of pH on condensed tannin (CT) efficacy to aggregate BSA.
ER50 is the half maximal effective ratio (values were corrected for CT content): 7.3 (±0.2) at pH 3, 1.0 (±0.0) at pH 4, 2.1 (±0.0) at pH 5, 3.4 (±0.0) at pH 6 (for citrate and BIS-TRIS buffer), 9.8 (±0.3) at pH 7, and no aggregation was observed at pH 8. The values in parentheses and error bars indicate the estimated error of the fit of the titration data for ER50 (after averaging experimental data points, typically n = 3 replicates).
Fig 3.
Experimental turbidimetry data obtained by adding condensed tannins (CT, Tilia flower F2 sample) to BSA.
(A) estimation of half maximal effective ratio (ER50) by a single sigmoid fit; (B) controls (CT addition to buffer/BSA, buffer addition to BSA).
Fig 4.
Influence of condensed tannin (CT) characteristics on aggregation of BSA.
ER50 –half maximal effective ratio; mDP–mean degree of polymerisation; amw–average molecular weight of CT; PC–procyanidins; cis–cis-flavan-3-ols.,● –B-type CT, ▲ –B-type galloylated CT, ■ –B-type with A-type linkages. Values corrected for CT content; CT fractions of >50 g CT/100 g of fraction; error bars are depicted, for more detail see Table 2; (A, B, E, F) fitted to power function; (A) ER50 [CT]/[BSA] (M/M) versus mDP, R2 = 0.84 (r = -0.916; p<0.01; df = 27; and rs = -0.926; p<0.01; df = 27); (B) ER50 [CT]/[BSA] (M/M) versus amw (kDa), R2 = 0.86 (r = -0.925; p<0.01; df = 27; and rs = -0.925; p<0.01; df = 27); (C) ER50 [CT]/[BSA] (M/M) versus PC (%); (D) ER50 [CT]/[BSA] (M/M) versus cis (%); (E) ER50 [CT]/[BSA] (mg/mL)/(mg/mL) versus mDP, R2 = 0.44 (r = -0.664; p<0.01; df = 27; and rs = -0.526; p<0.01; df = 27); (F) ER50 [CT]/[BSA] (mg/mL)/(mg/mL) versus amw (kDa), R2 = 0.45 (r = -0.674; p<0.01; df = 27 and rs = -0.521; p<0.01; df = 27); (G) ER50 [CT]/[BSA] (mg/mL)/(mg/mL) versus PC (%); (H) ER50 [CT]/[BSA] (mg/mL)/(mg/mL) versus cis (%).
Table 2.
Half maximal effective ratio (ER50) from condensed tannins (CT)-protein aggregation studies by turbidimetry.
Fig 5.
Turbidimetry data from the condensed tannin-BSA study overlaid with published isothermal titration calorimetry (ITC) results: ● –turbidimetry data fitted to a power function, Δ –ref. [19], and ϒ –ref. [22]; Note: the stoichiometric number (n) from ITC was divided by 3; ER50 –half maximal effective ratio; mDP–mean degree of polymerisation; DP–degree of polymerisation of oligomers.
Fig 6.
Influence of condensed tannin (CT) characteristics on gelatin aggregation.
ER50 –half maximal effective ratio, mDP–mean degree of polymerisation, amw–calculated average molecular weight of CT, PC–procyanidins, cis–cis-flavan-3-ols; ● –B-type CT, ▲ –B-type galloylated CT. Values corrected for CT content; CT fractions of >30 g CT/100 g of fraction; all data points are shown on the graph (for more details see Table 2); (A, B, E, F) fitted to power function for one replicate; (A) ER50 [CT]/[gelatin] (M/M) versus mDP, R2 = 0.92 (r = -0.961; p<0.01; df = 12 and rs = -0.951 p<0.01; df = 12); (B) ER50 [CT]/[gelatin] (M/M) versus amw (kDa), R2 = 0.96 (r = -0.981; p<0.01; df = 12 and rs = -0.958; p<0.01; df = 12); (C) ER50 [CT]/[gelatin] (M/M) versus PC (%); (D) ER50 [CT]/[gelatin] (M/M) versus cis (%); (E) ER50 [CT]/[gelatin] (mg/mL)/(mg/mL) versus mDP, R2 = 0.77 (r = -0.861; p<0.01; df = 12 and rs = -0.854; p<0.01; df = 12); (F) ER50 [CT]/[gelatin] (mg/mL)/(mg/mL) versus amw (kDa), R2 = 0.83 (r = -0.897; p<0.01; df = 12 and rs = -0.879; p<0.01; df = 12); (G) ER50 [CT]/[gelatin] (mg/mL)/(mg/mL) versus PC (%); (H) ER50 [CT]/[gelatin] (mg/mL)/(mg/mL) versus cis (%).
Fig 7.
Condensed tannin (CT) interactions study with BSA by circular dichroism (CD).
(A) Normalised CD spectra from qBiC software of BSA (reference in dark blue) and of BSA upon interactions with CT fractions (see legend in B); (B) CD difference spectrum of BSA treated with CT fractions calculated by subtracting the ‘CT only’ CD spectra (CDCT) and the ‘BSA only’ CD spectra (CDBSA) from the spectrum recorded with the CT and BSA mix (CDBSA_CT): ΔCD = CDBSA_CT - (CDCT + CDBSA).
Table 3.
Changes in BSA secondary structure upon binding of condensed tannins (CT).
Table 4.
Estimated quenching parameters, Stern-Volmer constant (KSV), for the interactions of condensed tannins (CT) with BSA.
Fig 8.
Fluorescence quenching of tryptophan in BSA in relation to condensed tannin (CT) characteristics.
KSV−Stern-Volmer quenching constant, i.e. slope obtained from linear part of Stern-Volmer plot fitted to linear regression: F0/F versus [CT]; mDP–mean degree of polymerisation; amw–average molecular weight of CT; PC–procyanidins, cis–cis-flavan-3-ols; ● –B-type CT, ▲ –B-type galloylated CT. Values corrected for CT content, error bars indicate the standard deviation of n = 3 replicates (if n<3, all data points are shown; for more details see Table 4); (A) KSV (mM-1) versus mDP; (B) KSV (mM-1) versus amw (kDa); (C) KSV (mM-1) versus PC (%); (D) KSV (mM-1) versus cis (%), R2 = 0.39, (rs = 0.678; p<0.05; df = 9); (E) KSV [(mg/mL)-1] versus mDP; (F) KSV [(mg/mL)-1] versus amw (kDa); (G) KSV [(mg/mL)-1] versus PC (%), R2 = 0.81 (r = 0.899; p<0.01; df = 9); (H) KSV [(mg/mL)-1] versus cis (%), R2 = 0.34, (rs = 0.887; p<0.01; df = 9).