Author Contributions
Conceptualization, F.J.L. and R.S.; methodology, A.E., F.J.L. and R.S.; formal analysis, F.J.L., A.E. and R.S.; investigation, I.Á.-B., A.V., A.B.C., M.A.B. and A.E.; resources, P.G., F.J.L. and R.S.; writing—original draft preparation, P.G., F.J.L. and R.S.; writing—review and editing, P.G., F.J.L. and R.S.; supervision, P.G., F.J.L. and R.S.; project administration, P.G., F.J.L. and R.S.; funding acquisition, P.G., F.J.L. and R.S. All authors have read and agreed to the published version of the manuscript.
Abbreviations
Aβ, amyloid β-peptide; AchE, acetylcholinesterase; ATC, acetylcholine; AD, Alzheimer’s disease; Api, apigenin; ATR, attenuated total reflectance; DLS, dynamic light scattering; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid; FTIR, Fourier-transform infrared; IPTG, 1-thio-β-D-galactopyranoside; MD, molecular dynamics; NB, native buffer; Quer, quercetin; RSC, radical scavenging capacity; RMSD, root-mean-square deviation; SAR, structure–activity relationships; MM-GBSA, molecular mechanics–generalized Born solvent area; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PDI, polydispersity; TEM, transmission electron microscopy; Th-T, thioflavin-T.
Figure 1.
Representations of the chemical structures of apigenin and quercetin.
Figure 1.
Representations of the chemical structures of apigenin and quercetin.
Figure 2.
ThT fluorescence in the presence of Aβ40: Effect of aggregation inhibitors. The samples were incubated at 37 °C for 24 h and fluorescence emission spectra were recorded in the range 465—600 nm. Green spectrum: typical aggregation of Aβ; black dotted spectrum: Aβ monomers incubated at 4 °C (negative control); red spectrum: aggregation in the presence of quercetin; blue spectrum: aggregation in the presence of apigenin. Conditions: [Quercetin/Apigenin] = 20 µM; [Aβ40] = 20 µM. The assays were performed in triplicates, and SD values <5% were obtained in all cases (not shown for the sake of clarity).
Figure 2.
ThT fluorescence in the presence of Aβ40: Effect of aggregation inhibitors. The samples were incubated at 37 °C for 24 h and fluorescence emission spectra were recorded in the range 465—600 nm. Green spectrum: typical aggregation of Aβ; black dotted spectrum: Aβ monomers incubated at 4 °C (negative control); red spectrum: aggregation in the presence of quercetin; blue spectrum: aggregation in the presence of apigenin. Conditions: [Quercetin/Apigenin] = 20 µM; [Aβ40] = 20 µM. The assays were performed in triplicates, and SD values <5% were obtained in all cases (not shown for the sake of clarity).
Figure 3.
Time-course kinetics of Aβ40 aggregation in the absence and presence of quercetin and apigenin, followed by ThT fluorescence. Green: without flavonoid; blue: with apigenin; red: with quercetin. Conditions: [Quercetin/Apigenin] = 20 µM; [Aβ40] = 20 µM.
Figure 3.
Time-course kinetics of Aβ40 aggregation in the absence and presence of quercetin and apigenin, followed by ThT fluorescence. Green: without flavonoid; blue: with apigenin; red: with quercetin. Conditions: [Quercetin/Apigenin] = 20 µM; [Aβ40] = 20 µM.
Figure 4.
Effect of quercetin and apigenin on the AChE-induced fibrillation of Aβ40. The samples were incubated at 37 °C for 24 h and ThT fluorescence emission spectra were recorded in the range 465–600 nm. Green spectrum: typical aggregation of Aβ; black dotted spectrum: Aβ monomers incubated at 4 °C (negative control); red spectrum: aggregation in the presence of quercetin; blue spectrum: aggregation in the presence of apigenin. Conditions: [Quercetin/Apigenin] = 20 µM; [Aβ40] = 20 µM; [AChE] = 4 μM. The assays were performed in triplicates, and SD values <5% were obtained in all cases (not shown for the sake of clarity).
Figure 4.
Effect of quercetin and apigenin on the AChE-induced fibrillation of Aβ40. The samples were incubated at 37 °C for 24 h and ThT fluorescence emission spectra were recorded in the range 465–600 nm. Green spectrum: typical aggregation of Aβ; black dotted spectrum: Aβ monomers incubated at 4 °C (negative control); red spectrum: aggregation in the presence of quercetin; blue spectrum: aggregation in the presence of apigenin. Conditions: [Quercetin/Apigenin] = 20 µM; [Aβ40] = 20 µM; [AChE] = 4 μM. The assays were performed in triplicates, and SD values <5% were obtained in all cases (not shown for the sake of clarity).
Figure 5.
Time-course kinetics of AChE-induced Aβ40 aggregation in the absence and presence of the two flavonoids quercetin and apigenin, followed by ThT fluorescence. Green: without flavonoid; blue: with apigenin; red: with quercetin. Conditions: [Quercetin/Apigenin] = 20 µM; [Aβ40] = 20 µM.
Figure 5.
Time-course kinetics of AChE-induced Aβ40 aggregation in the absence and presence of the two flavonoids quercetin and apigenin, followed by ThT fluorescence. Green: without flavonoid; blue: with apigenin; red: with quercetin. Conditions: [Quercetin/Apigenin] = 20 µM; [Aβ40] = 20 µM.
Figure 6.
TEM images of Aβ40 fibrils generated without (left) and with (right) AChE, and in the absence (top) or presence of the flavonoids apigenin (Api; middle) and quercetin (Quer; bottom). Scale bars: 200 nm.
Figure 6.
TEM images of Aβ40 fibrils generated without (left) and with (right) AChE, and in the absence (top) or presence of the flavonoids apigenin (Api; middle) and quercetin (Quer; bottom). Scale bars: 200 nm.
Figure 7.
FTIR patterns centered in the amide I region of Aβ40 for different samples after 24 h of incubation. Black spectrum: free Aβ; green spectrum: Aβ + apigenin; dark-blue spectrum: Aβ + quercetin; red spectrum: Aβ + AChE; light-blue spectrum: Aβ + AChE + apigenin; orange spectrum: Aβ + AChE + quercetin.
Figure 7.
FTIR patterns centered in the amide I region of Aβ40 for different samples after 24 h of incubation. Black spectrum: free Aβ; green spectrum: Aβ + apigenin; dark-blue spectrum: Aβ + quercetin; red spectrum: Aβ + AChE; light-blue spectrum: Aβ + AChE + apigenin; orange spectrum: Aβ + AChE + quercetin.
Figure 8.
AChE activities as a function of the concentration of flavonoid used, measured spectrophotometrically at 412 nm. The concentrations of apigenin (Api) and quercetin (Quer) flavonoids, i.e., [Drug], range from 0 to 50 μM; [AChE] = 0.08 U mL−1.
Figure 8.
AChE activities as a function of the concentration of flavonoid used, measured spectrophotometrically at 412 nm. The concentrations of apigenin (Api) and quercetin (Quer) flavonoids, i.e., [Drug], range from 0 to 50 μM; [AChE] = 0.08 U mL−1.
Figure 9.
Lineweaver–Buck plots. (a) Apigenin. (b) Quercetin.
Figure 9.
Lineweaver–Buck plots. (a) Apigenin. (b) Quercetin.
Figure 10.
Fluorescence quenching of AChE by quercetin and apigenin (Drug in the plots). (a) Intrinsic fluorescence emission of AChE upon addition of increasing amounts of quercetin (red) and apigenin (blue). (b) Stern–Volmer plots, viz. F0/F vs. [flavonoid] showing the quenching of AChE fluorescence in the presence of increasing amounts of quercetin (red) and apigenin (blue). (c) Relationship between the binding affinities and the numbers of binding sites for the interaction between AChE and apigenin (blue) and between AChE and quercetin (red). λexc = 275 nm, λem = 332 nm.
Figure 10.
Fluorescence quenching of AChE by quercetin and apigenin (Drug in the plots). (a) Intrinsic fluorescence emission of AChE upon addition of increasing amounts of quercetin (red) and apigenin (blue). (b) Stern–Volmer plots, viz. F0/F vs. [flavonoid] showing the quenching of AChE fluorescence in the presence of increasing amounts of quercetin (red) and apigenin (blue). (c) Relationship between the binding affinities and the numbers of binding sites for the interaction between AChE and apigenin (blue) and between AChE and quercetin (red). λexc = 275 nm, λem = 332 nm.
Figure 11.
Five selected binding modes for quercetin (carbon atoms in green) with different human AChE systems. (a) 6CQU—Replica 1 (carbon atoms in magenta), (b) 6CQU—Replica 2 (carbon atoms in orange), (c) 4M0F—Replica 2 (carbon atoms in light blue), (d) 4M0F—Replica 3 (carbon atoms in yellow) and (e) 6O4X—Replica 3 (carbon atoms in cyan). Selected residues in the binding pocket are indicated as bold text. Dashed lines represent interatomic distance (values in Å).
Figure 11.
Five selected binding modes for quercetin (carbon atoms in green) with different human AChE systems. (a) 6CQU—Replica 1 (carbon atoms in magenta), (b) 6CQU—Replica 2 (carbon atoms in orange), (c) 4M0F—Replica 2 (carbon atoms in light blue), (d) 4M0F—Replica 3 (carbon atoms in yellow) and (e) 6O4X—Replica 3 (carbon atoms in cyan). Selected residues in the binding pocket are indicated as bold text. Dashed lines represent interatomic distance (values in Å).
Figure 12.
Radical scavenging abilities of (a) apigenin and (b) quercetin, determined through the absorbance of DPPH at 517 nm, using increasing concentrations of the flavonoids.
Figure 12.
Radical scavenging abilities of (a) apigenin and (b) quercetin, determined through the absorbance of DPPH at 517 nm, using increasing concentrations of the flavonoids.
Table 1.
Kinetic parameters obtained for the aggregation of free Aβ40, and upon incubation with apigenin and quercetin.
Table 1.
Kinetic parameters obtained for the aggregation of free Aβ40, and upon incubation with apigenin and quercetin.
| Aβ40 | Aβ40 + Api | Aβ40 + Quer |
---|
kn (10−5 s−1) | 1.83 | 5.88 | 8.27 |
ke (M−1·s−1) | 175.73 | 191.27 | 80.93 |
t0 (s) | 1060.4 | 641.0 | 321.2 |
t1/2 (s) | 1844.1 | 1315.3 | 2182.1 |
t1 (s) | 2627.7 | 1989.6 | 4043.0 |
Inhibition (%) | 0.0 | 66.5 | 74.9 |
Table 2.
Kinetic parameters obtained for the AChE-induced aggregation of free Aβ40, Aβ40 with apigenin and Aβ40 with quercetin.
Table 2.
Kinetic parameters obtained for the AChE-induced aggregation of free Aβ40, Aβ40 with apigenin and Aβ40 with quercetin.
| Aβ40 + AChE | Aβ40 + AChE + Api | Aβ40 + AChE + Quer |
---|
kn (10−5 s−1) | 0.80 | 2.57 | 1.92 |
ke (M−1·s−1) | 309.0 | 246.2 | 333.7 |
t0 (s) | 868.6 | 736.8 | 481.9 |
t1/2 (s) | 1360.0 | 1323.2 | 1096.2 |
t1 (s) | 1851.3 | 1909.6 | 1710.6 |
Inhibition (%) | 0.0 | 25.5 | 57.9 |
Table 3.
Size distributions and polydispersity (PDI) of Aβ40 fibrils generated without and with AChE, and in the absence or presence of apigenin and quercetin, determined by dynamic light scattering (DLS).
Table 3.
Size distributions and polydispersity (PDI) of Aβ40 fibrils generated without and with AChE, and in the absence or presence of apigenin and quercetin, determined by dynamic light scattering (DLS).
| z-Average dm./nm | PDI |
---|
Aβ40 | 3074 | 0.59 |
Aβ40 + Apigenin | 2399 | 0.56 |
Aβ40 + Quercetin | 1402 | 0.46 |
Aβ40AChE | 1547 | 0.42 |
Aβ40AChE + Apigenin | 1585 | 0.58 |
Aβ40AChE + Quercetin | 1351 | 0.60 |
Table 4.
Absorption peaks characterizing the secondary structure of Aβ40, determined by FTIR spectroscopy.
Table 4.
Absorption peaks characterizing the secondary structure of Aβ40, determined by FTIR spectroscopy.
Peak | Peak (cm−1) | Area (%) |
---|
1 | 1611.1 | 31.4 |
2 | 1631.0 | 24.6 |
3 | 1650.1 | 6.1 |
4 | 1668.1 | 37.8 |
Table 5.
Binding affinities (Ka) and numbers of binding sites (n) for the interaction of apigenin and quercetin with AChE.
Table 5.
Binding affinities (Ka) and numbers of binding sites (n) for the interaction of apigenin and quercetin with AChE.
| AChE + Apigenin | AChE + Quercetin |
---|
Ka (106 M−1) | 0.53 | 0.13 |
n | 1.09 | 1.01 |
Table 6.
Binding affinities (Ka) and numbers of binding sites (n) for the interaction of apigenin and quercetin with AChE.
Table 6.
Binding affinities (Ka) and numbers of binding sites (n) for the interaction of apigenin and quercetin with AChE.
Models | ΔEvw | ΔEel | ΔGsol,el | ΔGsol-n-el | ΔGbind |
---|
6CQU—Replica 1 | −35.2 ± 2.1 | −41.7 ± 4.9 | −41.7 ± 4.9 | −41.7 ± 4.9 | −21.0 ± 2.5 |
6CQU—Replica 2 | −36.0 ± 2.2 | −25.8 ± 4.3 | −25.8 ± 4.3 | −25.8 ± 4.3 | −19.6 ± 2.8 |
4M0F—Replica 2 | −34.0 ± 2.2 | −24.7 ± 6.8 | −24.7 ± 6.8 | −24.7 ± 6.8 | −17.2 ± 3.0 |
4M0F—Replica 3 | −37.1 ± 1.9 | −25.9 ± 4.3 | −25.9 ± 4.3 | −25.9 ± 4.3 | −21.3 ± 2.5 |
6O4X—Replica 3 | −33.8 ± 2.6 | −44.6 ± 8.3 | −44.6 ± 8.3 | −44.6 ± 8.3 | −16.6 ± 2.9 |
Table 7.
Radical scavenging capacity (RSC, in %) of apigenin and quercetin, determined by their ability to quench the absorbance of the DPPH radical.
Table 7.
Radical scavenging capacity (RSC, in %) of apigenin and quercetin, determined by their ability to quench the absorbance of the DPPH radical.
[Quercetin/Apigenin]/μM | Apigenin RSC (%) | Quercetin RSC (%) |
---|
1 | −0.1 | 30.2 |
2 | 1.8 | 65.1 |
3 | 0.2 | 99.3 |
4 | 2.4 | 100.9 |
10 | −0.7 | 100.1 |
100 | 2.9 | 99.7 |