Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online
Journal website www.isnff-jfb.com

Original Research

Volume 14, June 2021, pages 102-113

A novel approach to trypsin inhibition by flavonoids

Figures

Figure 1.
Figure 1. Chemical structures of evaluated flavonoids: (a) Hesperetin (HES), (b) luteolin (LUT), (c) quercetin (QUE), (d) catechin (CAT), and (e) rutin (RUT).
Figure 2.
Figure 2. Quenching of intrinsic trypsin fluorescence in the presence of different concentrations of flavonoids (3.0–100.0 µM). (a) HES, (b) LUT, (c) QUE, (d) CAT, and (e) RUT. Fluorescence was recorded after 60 min of incubation with the flavonoid, at an excitation wavelength of 290 nm. Data represent the average of three experimental replicates.
Figure 3.
Figure 3. Fluorescence spectra of trypsin-ANS complex in the absence and presence of different concentrations (up to 300 µM) of flavonoids: (a) HES, (b) LUT, (c) QUE, (d) CAT, and (e) RUT. Insert shows plot of flavonoid concentration (mM) versus corrected fluorescence intensity change DF. Samples were excited at a wavelength of 290 nm. Yellow arrows indicate the trend of fluorescence change as the concentration of flavonoids increases.
Figure 4.
Figure 4. Circular dichroism absorption spectra (CD) of trypsin in the presence of different concentrations (10–100 µM) flavonoids: (a)HES, (b) LUT, (c) QUE, (d) CAT, and (e) RUT. Absorption wavelength (λ), and degrees of ellipticity, are in nanometers (nm), and in milligrade ellipticity (mdeg), respectively. Yellow arrows indicate changes in the spectrum (increase or decrease), as the concentration of flavonoids increases.
Figure 5.
Figure 5. Molecular docking of trypsin and potential binding sites for the flavonoids HES, LUT, QUE, CAT, RUT, and the substrate, BAPNA. Surface representation for the full tridimensional structure of trypsin (dark gray), the amino acids of the active site are represented in yellow and the possible binding sites for the ligands (a). Flavonoids binding sites and the main amino acids residues (sticks) are shown (b), as well as the main for the flavonoids and the main specific interactions for HES (c), LUT (d), QUE (e), CAT (f), and RUT (g) are noted. The dot lines and distances (Å) with colors correspond to different interactions such as hydrogen (red), and hydrophobic binding(green).

Tables

Table 1. Trypsin apparent catalytic parameters (V appmax, KappM, Ki and Ki′) and inhibition type for the hydrolysis of BAPNA in the presence of the flavonoids
 
FlavonoidConcentration (μM)V appmax (10−2 mM/min)KappM (mM)Inhibition typeKi and K′i (mM)
Data are represented as mean value ± standard deviation of triplicate analysis. Different letters in the same column indicate statistically significant values (Fisher's least significant difference analysis, p < 0.05) respect to control, or between treatments for free enzyme or enzyme substrate dissociation constants (Ki and Ki′, respectively). Vmax and KM correspond to maximal rate and Michaelis-Menten constant, respectively. n.d. means not determined.
CONTROL0.003.75 ± 0.20a0.35 ± 0.05fgNone0.000.00
HES12.553.60 ± 0.10a0.35 ± 0.01gMixed73.15 ± 1.90a78.05 ± 2.50a
25.103.60 ± 0.05a0.40 ± 0.05fg
50.093.40 ± 0.70b1.00 ± 0.10d
LUT12.703.05 ± 0.50ab1.00 ± 0.00dMixed42.05 ± 0.70d44.50 ± 0.85c
25.452.10 ± 0.00e2.40 ± 0.15b
50.201.40 ± 0.70f5.60 ± 0.95a
QUE12.563.60 ± 0.15a0.40 ± 0.00fMixed58.90 ± 0.10c61.00 ± 1.30b
25.083.40 ± 0.10b0.80 ± 0.18d
50.322.65 ± 0.20d1.25 ± 0.05c
CAT12.493.80 ± 0.25a0.35 ± 0.00gNonen.d.n.d.
25.073.70 ± 0.00a0.35 ± 0.00g
50.913.70 ± 0.15a0.35 ± 0.05g
RUT12.563.70 ± 0.05a0.40 ± 0.10fgMixed68.20 ± 2.10b75.00 ± 8.00a
25.003.40 ± 0.20a0.50 ± 0.00e
50.543.00 ± 0.05c1.05 ± 0.30cd

 

Table 2. Fluorescence quenching parameters (KappD, Ksv, kq, n and Ka) of trypsin in the presence of flavonoids
 
FlavonoidKappD (µM)Ksv (10−1 mM−1)kq (10−12 mM−1 s−1)nKa (10−1 mM−1)
Data are the mean value ± standard deviation of triplicate analysis. Different letters in the same column indicate statistically significant values (Fisher's least significant difference analysis, p < 0.05) respect to control. n.d. means not determined.
HES38.00 ± 4.02a0.96 ± 0.05b0.10 ± 0.01c0.70 ± 0.15b0.60 ± 0.00d
LUT16.10 ± 3.00c1.87 ± 0.10a9.86 ± 1.05a0.90 ± 0.01a1.60 ± 0.22a
QUE17.50 ± 1.50c1.22 ± 0.30b6.43 ± 0.40b0.80 ± 0.20ab0.90 ± 0.14b
CATn.d.0.00 ± 0.00d0.00 ± 0.00d0.00 ± 0.00d0.00 ± 0.00e
RUT31.00 ± 2.67b0.51 ± 0.00c6.61 ± 0.15b0.60 ± 0.03c0.70 ± 0.01c

 

Table 3. Gibbs free energy (ΔG°, kCal/mol), the trypsin amino acid residues interacting with flavonoids, and the distance (Å) for the possible enzyme-flavonoid conformations
 
FlavonoidsAmino acid residues lining the binding site, distance and binding energy interaction
HydrophobicHydrogen BindingΔG°
HESTyr151 (3.6; 3.8)Asp74 (2.8), Tyr151 (3.2)−6.7
LUTTyr151 (3.8; 3.7; 3.7; 3.8)Asp74 (3.1), Gln192 (2.4)−7.3
QUETyr151 (3.9; 3.7; 4.0; 3.9)Gly193 (3.1)−6.5
CATNingunaAsn34 (3.1), Arg62 (2.8), Gln64 (3.1)−7.5
RUTNingunaAsn97 (2.9; 3.1), Ser195 (2.8), Gln192 (2.7), Gly216 (2.7)−7.0