Site-specific Inhibitory Mechanism for Amyloid β42 Aggregation by Catechol-type Flavonoids Targeting the Lys Residues

Background: The inhibitory mechanism of Aβ42 aggregation by flavonoid is fully unknown.

Results: The oxidant enhanced the inhibitory activity of (+)-taxifolin against Aβ42 aggregation by forming Aβ42-taxifolin adducts between the Lys residues and oxidized (+)-taxifolin.

Conclusion: The inhibitory activity of catechol-type flavonoids requires autoxidation to form an o-quinone to react with Lys.

Significance: These may help design promising inhibitors against Aβ42 aggregation for Alzheimer disease therapy.

Abstract

The aggregation of the 42-residue amyloid β-protein (Aβ42) is involved in the pathogenesis of Alzheimer disease (AD). Numerous flavonoids exhibit inhibitory activity against Aβ42 aggregation, but their mechanism remains unclear in the molecular level. Here we propose the site-specific inhibitory mechanism of (+)-taxifolin, a catechol-type flavonoid, whose 3′,4′-dihydroxyl groups of the B-ring plays a critical role. Addition of sodium periodate, an oxidant, strengthened suppression of Aβ42 aggregation by (+)-taxifolin, whereas no inhibition was observed under anaerobic conditions, suggesting the inhibition to be associated with the oxidation to form o-quinone. Because formation of the Aβ42-taxifolin adduct was suggested by mass spectrometry, Aβ42 mutants substituted at Arg⁵, Lys¹⁶, and/or Lys²⁸ with norleucine (Nle) were prepared to identify the residues involved in the conjugate formation. (+)-Taxifolin did not suppress the aggregation of Aβ42 mutants at Lys¹⁶ and/or Lys²⁸ except for the mutant at Arg⁵. In addition, the aggregation of Aβ42 was inhibited by other catechol-type flavonoids, whereas that of K16Nle-Aβ42 was not. In contrast, some non-catechol-type flavonoids suppressed the aggregation of K16Nle-Aβ42 as well as Aβ42. Furthermore, interaction of (+)-taxifolin with the β-sheet region in Aβ42 was not observed using solid-state NMR unlike curcumin of the non-catechol-type. These results demonstrate that catechol-type flavonoids could specifically suppress Aβ42 aggregation by targeting Lys residues. Although the anti-AD activity of flavonoids has been ascribed to their antioxidative activity, the mechanism that the o-quinone reacts with Lys residues of Aβ42 might be more intrinsic. The Lys residues could be targets for Alzheimer disease therapy.

Introduction

Alzheimer disease (AD)³ is characterized by amyloid deposition in senile plaques mainly consisting of 40- and 42-mer amyloid β-proteins (Aβ40, Aβ42) (1, 2). These proteins are generated from the amyloid precursor protein by β- and γ-secretases (amyloidogenic pathway). Aβ aggregates mainly through intermolecular β-sheet formation and shows neurotoxicity in vitro (3). Aβ42 plays a more pivotal role in the pathogenesis of AD than Aβ40 because of its higher aggregative ability and neurotoxicity (3). It has been well documented that soluble Aβ oligomeric assemblies rather than insoluble fibrils cause memory loss and neuronal death (4, 5). Oxidative stress is one of the major contributing factors to neurodegenerative disease progression (6). Aβ-induced toxicity has been correlated to oxidative damage through protein radicalization in vitro (7, 8) and in vivo (9, 10).

Researchers have reported protective effects of various polyphenols from green tea, turmeric, and red wine etc., against Aβ aggregation and neurotoxicity (11–13). Several compounds (e.g. (−)-epigallocatechin-3-gallate (EGCG), curcumin, and resveratorol) are in clinical or preclinical trials for AD treatment (14, 15). However, the recent failures of some trials (16) motivated us to clarify the mechanism by which polyphenols inhibit the aggregation of Aβ42 to develop promising leads for clinical use.

Concerning the molecular interaction of Aβ with flavonoids, a docking simulation by Keshet et al. (17) predicted the involvement of Lys²⁸ and the C-terminal region in the binding with myricetin. However, the precise mode of binding with flavonoids has scarcely been addressed, except for limited studies using NMR spectroscopy (curcumin (18), EGCG (19), and myricetin (20)), which suggested less-specific interaction with the β-sheet region in Aβ.

Our group recently found that silymarin, seed extracts of Silybum marianum, attenuated AD-like pathologic features, such as senile plaques, neuroinflammation, behavioral dysfunction, and Aβ oligomer formation using a well established AD mouse model (J20) (21). We also isolated (+)-taxifolin (22), a flavanonol that has a catechol moiety on the B-ring (Fig. 1A), as a component of the extracts that prevents Aβ42 aggregation (23). “Aggregation” used in this article means the process of Aβ42 monomer to form fibrils by way of oligomer and/or protofibril. This article describes a comprehensive study on the ability of (+)-taxifolin to prevent aggregation and β-sheet formation of Aβ42, along with the effects of various flavanonols and flavonols on the aggregation of Aβ42 mutants substituted at Arg⁵, Lys¹⁶, and/or Lys²⁸ with norleucine (Nle). These results together with the results of liquid chromatography-mass spectrometry (LC-MS) led us to propose a site-specific inhibitory mechanism for Aβ42 aggregation by catechol-type flavonoids, where adduct formation at Lys residues in Aβ42 with the o-quione derived from flavonoids could be involved in the suppression of Aβ42 aggregation.

FIGURE 1.

Effects of autoxidation of (+)-taxifolin on its ability to inhibit Aβ42 aggregation and Met³⁵ oxidation. A, the structure of (+)-taxifolin. B, the effect of sodium periodate (NaIO₄), an oxidant, on Aβ42 aggregation estimated by Th-T tests. Aβ42 (25 μm) was incubated with or without (+)-taxifolin (50 μm) and/or NaIO₄ (100 μm) at 37 °C. C, the ability of (+)-taxifolin to suppress the aggregation of Aβ42 under an anaerobic condition. Aβ42 (25 μm) was incubated with or without (+)-taxifolin (50 μm) in vacuo at room temperature. D, HPLC analysis of Aβ42 solution with the indicated treatment. Aβ42 (25 μm) was incubated with or without (+)-taxifolin (50 μm) and/or NaIO₄ (100 μm) at 37 °C for 4 h. An aliquot was centrifuged by 20,130 × g at 4 °C for 10 min, and the supernatant was subjected to HPLC on a Develosil ODS UG-5 column under a gradient of 10–50% CH₃CN containing 0.1% NH₄OH for 40 min. E, the effect of TCEP-HCl (denoted as TCEP), a reductant, on Aβ42 aggregation. Aβ42 (25 μm) was incubated with or without (+)-taxioflin (50 μm) and/or TCEP (100 μm) at 37 °C. F, the aggregative ability of Aβ42 in the presence of Aβ42 seed and/or (+)-taxifolin, NaIO₄. Aβ42 (25 μm) was incubated with or without the seed (10 μg/ml) and/or (+)-taxifolin (50 μm) or NaIO₄ (100 μm) at 37 °C. The data are presented as the mean ± S.E. (n = 8). Th-T relative fluorescence was expressed as a percentage of the fluorescence for wild-type Aβ42 alone, whose maximum value was taken as 100%.

EXPERIMENTAL PROCEDURES

Synthesis of (+)-Taxifolin, Dihydrokaempferol, and Pinobanksin

A naturally occurring form of (+)-taxifolin was synthesized (supplemental Scheme S1A) basically according to the previous reports (23, 24). Briefly, vanillin was demethylated by treatment with boron tribromide to give 3,4-dihydroxybenzaldehyde quantitatively, whose phenolic hydroxyl groups were protected with methoxymethyl groups. The phenolic hydroxyl groups of 2,4,6-trihydroxyacetophenone were also protected with methoxymethyl groups. A cross-aldol reaction between these products, followed by treatment with H₂O₂ under a basic condition, yielded the epoxide 7 (supplemental Scheme S1A), which was cyclized and deprotected under an acidic condition to give (±)-taxifolin. (+)-Taxifolin was separated by HPLC on a CHIRALCEL OJ-RH column (10 mm inner diameter × 150 mm; Daicel Corporation, Osaka, Japan) using 15% CH₃CN/H₂O containing 0.1% acetic acid (25). Synthesis of ¹³C₆-(+)-taxifolin was performed by using vanillin ring-¹³C₆ (Isotec, Miamisburg, OH) as a starting material.

(±)-Dihydrokaempferol (26) and (±)-pinobanksin (27) were synthesized in a manner similar to (±)-taxifolin using 4-hydroxybenzaldehyde or benzaldehyde in place of vanillin as a starting material, respectively (supplemental Scheme S1A). Enantiomers were not separated because the inhibitory effect on Aβ42 aggregation by (−)-taxifolin was almost the same as that by (+)-taxifolin (23). The structure of each of these compounds was confirmed by ¹H NMR (AVANCE III 500; TMS, Bruker, Germany) and EI-MS (JMS-600H; JEOL, Tokyo, Japan). EI-MS data were as follows: (+)-taxifolin (m/z 304 [M]⁺), ¹³C₆-(+)-taxifolin (m/z 310 [M]⁺), dihydrokaempferol (m/z 288 [M]⁺), and pinobanksin (m/z 272 [M]⁺). The spectra of ¹H NMR (28) and ¹³C NMR (29) of ¹³C₆-(+)-taxifolin are shown in supplemental Fig. S1. The optical rotation of each enantiomer was (+)-taxifolin [α]_D +17.3 (c 0.1, MeOH), (−)-taxifolin [α]_D −16.2 (c 0.1, MeOH), almost equal to those reported previously; (+)-taxifolin [α]_D +19.0 (c 0.1, MeOH) (22). Other flavonoids: myricetin (Wako, Osaka, Japan), kaempferol, (±)-dihydromyricetin (ChromaDex, Irvine, CA), morin, galangin, quercetin (Sigma), and datiscetin (Extrasynthese, Genay, France) were purchased commercially.

Trapping of the o-Quinone Form of (+)-Taxifolin by Phenylenediamine

Sodium periodate (NaIO₄, 19 mg, 89 μmol) in H₂O (0.20 ml) was added to (±)-taxifolin (28 mg, 91 μmol; Toronto Research Chemicals Inc., North York, ON, Canada) in methanol (3.5 ml). After stirring for 15 min at room temperature, the reaction mixture was extracted with ethyl acetate (5.0 ml), to which 1,2-phenylenediamine (9.8 mg, 91 μmol; Wako) was added before stirring for 30 min at room temperature. The mixture was concentrated and separated by HPLC on a YMC SH-342–5AL column (20 mm inner diameter × 150 mm; YMC, Kyoto, Japan) with 60% MeOH/H₂O to give the corresponding phenazine (3.8%) (supplemental Scheme S1B). The structure was confirmed by ¹H NMR and high resolution EI-MS: ¹H NMR (500 MHz, 295.3 K, acetone-d₆, 6.98 mm) δ 4.87 (1H, d, J = 11.5 Hz), 5.59 (1H, d, J = 11.5 Hz), 6.07 (1H, s), 6.09 (1H, s), 7.96–8.00 (2H, m), 8.22 (1H, dd, J = 9.0, 1.7 Hz), 8.26–8.30 (2H, m), 8.33 (1H, d, J = 9.0 Hz), 8.46 (1H, d, J = 1.7 Hz), 11.72 (1H, brs); high resolution EI-MS m/z 374.0902 [M]⁺, calculated for C₂₁H₁₄N₂O₅ 374.0903.

Thioflavin-T Fluorescence Assay

The aggregative ability of Aβ42 was evaluated at 37 °C by the thioflavin-T (Th-T) method developed by Naiki and Gejyo (30). The procedure was described elsewhere (31). Fluorescence intensity was measured at 420 nm excitation and 485 nm emission using a microplate reader (MPR-A4ιII; TOSOH, Tokyo, Japan, or Fluoroskan Ascent; Thermo Scientific, Rockford, IL). In brief, Aβ42 was dissolved in 0.1% NH₄OH at 250 μm, and each flavonoid was dissolved in EtOH at 5 mm, followed by dilution with sodium phosphate-buffered saline (PBS: 50 mm sodium phosphate and 100 mm NaCl, pH 7.4) at the desired concentration (Aβ42, 25 μm; flavonoids, 50 μm). NaIO₄ or Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) was initially dissolved in PBS at 100 mm, then diluted with PBS at 100 μm before use. Experiments under an anaerobic condition were performed in a desiccator evacuated by a diaphragm pump (about 8 mm Hg; KNF Lab LABOPORT vacuum pump, KNF Neuberger, NJ) at room temperature. Unless otherwise noted, the concentrations of Aβ42, flavonoids, and oxidant/reductant used in this study were 25, 50, and 100 μm, respectively.

The effect of the addition of NaIO₄ on Met³⁵ oxidation was estimated by HPLC on a Develosil ODS UG-5 column (6.0 mm inner diameter × 100 mm; Nomura Chemical, Seto, Japan) under a gradient of 10–50% CH₃CN containing 0.1% NH₄OH for 40 min after the centrifugation of the Aβ42 solution at 20,130 × g at 4 °C (MX-300; TOMY, Tokyo, Japan) for 10 min.

The seeds of Aβ42 were also prepared basically according to the protocol developed by Naiki and Gejyo (30). Briefly, after incubation of Aβ42 (25 μm) in PBS (pH 7.4) for 24 h at 37 °C, the pellet obtained by centrifugation at 20,130 × g at 4 °C for 1 h was suspended by pipetting in PBS (pH 7.4) at a concentration of 1 mg/ml. The resultant solution was sonicated for 1 h in an ultrasonic device (MUS-20; EYELA, Tokyo, Japan), followed by dilution with PBS at 10 μg/ml before use. Th-T relative fluorescence was expressed as a percentage of wild-type Aβ42 alone, whose maximum value was taken as 100%.

Transmission Electron Microscopy (TEM)

The aggregates of Aβ42 after a 48-h incubation were examined under a H-7650 electron microscope (Hitachi, Ibaraki, Japan). The experimental procedure was described elsewhere (31).

UV-visible Spectrometry

Oxidation of (+)-taxifolin was monitored by UV spectroscopy (UV-2200A; Shimadzu, Kyoto, Japan). (+)-Taxifolin (50 μm) was incubated with Aβ42 (25 μm) in PBS (50 mm sodium phosphate and 100 mm NaCl, pH 7.4) at 37 °C. The solution was then loaded into a 1-cm path length quartz cell, and UV spectra were recorded at 200–500 nm. The sample was diluted three times with PBS because of its strong absorbance.

Circular Dichroism (CD) Spectrometry

The secondary structure of Aβ42 was estimated by CD spectrometry (J-805; JASCO, Tokyo, Japan) using a 0.1-mm quartz cell (121.027-QS, φ 10 mm; JASCO), as described elsewhere (32). Aβ42 (25 μm) was incubated with or without (+)-taxifolin (50 μm) in PBS (50 mm sodium phosphate and 100 mm NaCl, pH 7.4) at 37 °C. An aliquot was loaded into the quartz cell, and CD spectra were recorded at 190–250 nm. Experiments under an anaerobic condition were performed as described before. The spectra of Aβ42 mutants are shown after subtraction of the spectrum for vehicle alone, and those in the presence of (+)-taxifolin are shown after subtraction of the spectrum for (+)-taxifolin alone.

LC-MS Analysis

Aβ42 solution (25 μm) was incubated with 50 μm (+)-taxifolin in PBS (50 mm sodium phosphate and 100 mm NaCl, pH 7.4) in the presence of 100 μm NaIO₄ at 37 °C. After a 4-h incubation, the mixture was desalted and condensed twice by ZipTip C18 (Millipore, Bedford, MA). Five microliters of the solution was subjected to a liquid chromatography mass spectrometry ion trap time-of-flight (LCMS-IT-TOF; Shimadzu) through a YMC-Pack ODS-AQ column (6.0 mm inner diameter × 100 mm; YMC) at 25 °C under a gradient of 20–60% CH₃CN containing 0.1% formic acid for 30 min.

Synthesis of Aβ42 Mutants

Fmoc-Nle-OH was purchased from Watanabe Chemical Industries (Hiroshima, Japan). l-Alanine (¹³C₃, ¹⁵N), l-phenylalanine (¹³C_β), and l-valine (¹³C₅, ¹⁵N) were purchased from Isotec, and l-lysine (¹³C₆, ¹⁵N₂) and l-serine (¹³C₃, ¹⁵N) from Cambridge Isotope Laboratories (Andover, MA). Each labeled amino acid was protected by an Fmoc group as previously reported (33, 34). The structure of each Fmoc derivative was confirmed by ¹H NMR, ¹³C NMR, and fast atom bombardment-MS.

The Aβ42 mutants were synthesized in a stepwise fashion on 0.1 mmol of preloaded Fmoc-l-Ala-PEG-PS resin using a Pioneer^TM Peptide Synthesizer (Applied Biosystems, Foster City, CA) as reported previously (35). After the chain elongation was completed, the peptide-resin was treated with a mixture containing trifluoroacetic acid, m-cresol, thioanisol, and 1,2-ethanedithiol for final deprotection and cleavage from the resin. The crude peptide was precipitated by diethyl ether and purified by HPLC under an alkaline condition as described previously (31). After lyophilization, we obtained the corresponding pure Aβ42 peptide, the purity of which was confirmed by HPLC (>98%). The molecular weight of each Aβ42 mutant was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS, AXIMA-CFR; Shimadzu); R5Nle-Aβ42 (m/z, calculated: 4472.11; found: 4472.38 [M + H]⁺), K16Nle-Aβ42 (m/z, calculated: 4500.12; found: 4500.25 [M + H]⁺), K28Nle-Aβ42 (m/z, calculated: 4500.12; found: 4500.32 [M + H]⁺), K16,K28(Nle)₂-Aβ42 (m/z, calculated: 4485.11; found: 4485.13 [M + H]⁺), R5,K16,K28(Nle)₃-Aβ42 (m/z, calculated: 4442.08; found: 4442.58 [M + H]⁺), ¹³C,¹⁵N labeled-Aβ42 (m/z, calculated: 4538.90; found: 4538.94 [M + H]⁺).

Solid-state NMR Analysis

Aβ42 was labeled at Ala² (¹³C₃, ¹⁵N), Ser⁸ (¹³C₃, ¹⁵N), Lys¹⁶ (¹³C₆, ¹⁵N₂), Val¹⁸ (¹³C₅, ¹⁵N), Phe¹⁹, and Phe²⁰ (¹³C_β). (+)-Taxifolin was labeled with ¹³C₆ on the B-ring as mentioned above (23, 24). The labeled Aβ42 (13 μm) was incubated with ¹³C₆-(+)-taxifolin (145 μm) in PBS (50 mm sodium phosphate and 100 mm NaCl, pH 7.4) at 37 °C. After 48 h of incubation, the solution was centrifuged (27,720 × g, PRP-20–2; Hitachi) for 15 min at 4 °C, and then the precipitate was dried in vacuo to give the labeled Aβ42 aggregate associated with ¹³C₆-(+)-taxifolin (12 mg). The solid-state NMR experiments were performed at 14 T (600 MHz for ¹H) using a JEOL ECA-600 spectrometer and a custom-fabricated probe with a Chemagnetics 3.2-mm spinning system at a magic angle spinning frequency of 21 kHz at room temperature as reported previously (18). The ¹³C chemical shifts were calibrated in ppm relative to tetramethylsilane by considering the ¹³C chemical shift for methine ¹³C of solid adamantine (29.5 ppm) as an external reference. The ¹³C chemical shifts of labeled Aβ42 and (+)-taxifolin were assigned according to one-dimensional ¹³C CP/magic angle spinning NMR spectra (supplemental Fig. S2A). For the broadband ¹³C-¹³C correlation two-dimensional experiments, dipolar-assisted rotational resonance was used (36). Pulse sequence parameters of the NMR experiment were as follows: two pulse phase-modulated ¹H decoupling power = 80 kHz, RAMP-CP contact time = 1.2 ms, pulse delay = 2 s, t₁ increment = 23.7 μs, t₁ points of two-dimensional = 128 pt, and mixing time (τ_m) = 50 or 500 ms. The window function “HAMMING” was used in all two-dimensional FT spectra to minimize t₁ noise. As the two-dimensional FT dipolar-assisted rotational resonance spectra were difficult to analyze because of the t₁ noise (supplemental Fig. S2C), we applied covariance data processing to obtain a better representation of the two-dimensional spectrum (supplemental Fig. S2B). After Fourier transformation along the t₂ dimension and phase correction, the resulting data matrix was used for covariance processing as previously reported (18, 37). The covariance processing step was accelerated by singular value decomposition (38).

Statistical Analyses

All data are presented as the mean ± S.E. and the differences were analyzed with an one-way analysis of variance followed by Bonferroni's test or unpaired Student's t test. These tests were implemented within GraphPad Prism software (version 5.0d). p values < 0.05 were considered significant.

RESULTS

Effects of Autoxidation of (+)-Taxifolin on Its Ability to Prevent the Aggregation of Aβ42

We recently revealed that a catechol moiety on the B-ring of (+)-taxifolin (Fig. 1A) played an important role on the inhibitory activity against Aβ42 aggregation (23). A catechol moiety is easily oxidized to form an o-quinone (39). To investigate the contribution of autoxidation to the inhibitory ability, we examined the aggregative ability of Aβ42 in the presence of (+)-taxifolin treated with sodium periodate (NaIO₄), which is known as an oxidant of catechol (40). As shown in Fig. 1B, NaIO₄ extensively promoted the suppressive ability of (+)-taxifolin compared with (+)-taxifolin alone. These observations were also confirmed by the TEM experiment (Fig. 2A). NaIO₄ treatment in the presence of (+)-taxifolin formed only shorter and thinner fibrils compared with (+)-taxifolin alone. Aβ42 formed the typical fibrils even in the presence of NaIO₄ alone, and almost no differences (e.g. length, thickness) were observed between morphology in the presence and absence of NaIO₄ (Fig. 2A).

FIGURE 2.

Effects of autoxidation of (+)-taxifolin on the morphology of Aβ42 aggregates. A, TEM images of Aβ42 aggregates after 48 h of incubation. Aβ42 (25 μm) was treated with or without (+)-taxifolin (50 μm) and/or NaIO₄ (100 μm) under an aerobic or anaerobic condition. Scale bar = 200 nm. B, UV-visible spectra of (+)-taxifolin (50 μm) treated with Aβ42 (25 μm) in the presence of NaIO₄ (100 μm) or TCEP (100 μm) after incubation for 0, 4, 8, 24, and 48 h, respectively.

NaIO₄ alone slightly affected the Th-T fluorescence of Aβ42 aggregates (Fig. 1B) possibly because NaIO₄ can oxidize Met³⁵ in Aβ42 to its sulfoxide, the formation of which was confirmed by HPLC (Fig. 1D) and MALDI-TOF-MS (Aβ42-M35^ox; m/z: calculated: 4531.14; found: 4531.55 [M + H]⁺). This is in good agreement with a report that oxidation using hydrogen peroxide, a strong oxidant, reduced Aβ42 aggregation (41). However, in the presence of both (+)-taxifolin (50 μm) and NaIO₄ (100 μm), Met³⁵ was not oxidized by NaIO₄; this was confirmed by HPLC (Fig. 1D) and MALDI-TOF-MS (Aβ42-M35^red; m/z: calculated: 4515.14; found: 4516.26 [M + H]⁺). This indicates that NaIO₄ preferred to oxidize (+)-taxifolin more than the sulfur atom of the Met³⁵ of Aβ42.

In addition, we tested whether treatment of NaIO₄ leads to the oxidation of Met³⁵ in the preformed Aβ42 fibrils. The fibrils (about 28 μg) treated with NaIO₄ for 4 h were dissolved in formic acid (10 μl), and were sonicated for 1 h. After volatilization, the resultant pellets were resolved in 50% acetonitrile containing 0.1% trifluoroacetic acid, followed by subjection to MALDI-TOF-MS analysis. NaIO₄ did not oxidize Met³⁵ in the preformed Aβ42 fibril (Aβ42-M35^red; m/z: calculated: 4515.14; found: 4515.12 [M + H]⁺). Also in Th-T assay, Aβ42 fibril was not disassembled by NaIO₄ (data not shown). This means that NaIO₄ could partially oxidize Met³⁵ in the monomeric Aβ42, but not the fibrils.

To investigate the role of oxygen, suppression of Aβ42 aggregation by (+)-taxifolin was tested in vacuo. Notably, (+)-taxifolin little suppressed the aggregation of Aβ42 under an anaerobic condition (Fig. 1C). In TEM images, typical fibril formation was observed even in the presence of (+)-taxifolin under the anaerobic condition (Fig. 2A). Furthermore, Aβ42 aggregated in the presence of (+)-taxifolin and TCEP, a reductant (Fig. 1E). These results suggest the autoxidation of (+)-taxifolin is required for the inhibitory activity against Aβ42 aggregation.

The mechanism of Aβ42 fibril formation is well explained by a nucleation-dependent polymerization model mainly consisting of nucleation and extension phases (42, 43). To determine which stage (nucleation phase or extension phase) was affected by (+)-taxifolin, we examined the effect of (+)-taxifolin on Aβ42 aggregation in the presence of the fibril seed as a template, according to the protocol developed by Naiki and Gejyo (30). As shown in Fig. 1F, there was a nucleation phase (∼1 h) when Aβ42 was incubated alone, whereas addition of the seeds skipped the nucleation phase, resulting in rapid formation of Aβ42 fibrils. In the case of co-incubation of (+)-taxifolin with the seeds, the nucleation phase of Aβ42 did not drastically change, but fluorescence gradually decreased after incubation for 4 h, suggesting that (+)-taxifolin could prevent the elongation phase (∼2 or 4 h) in Aβ42 aggregation, rather than the nucleation phase (∼1 h) (Fig. 1B, F). Although the slight difference of the length of the elongation phase between Fig. 1, B and F, might be deduced from several factors, for example, outside temperature or batch (lot) of Aβ42, an averaged time of 2–4 h for the elongation phase was observed in another independent experiment. Moreover, we have recently reported the ability of (+)-taxifolin to destabilize the preformed Aβ42 fibril (23). The disappearance of the nucleation phase in the presence of seed and NaIO₄ (Fig. 1F) implied the ability of oxidized taxilfolin to disassemble even the seed. Indeed, the fluorescence of preformed Aβ42 fibrils immediately disappeared after addition of the (+)-taxifolin treated with NaIO₄ (data not shown).

Next, we measured UV spectra of (+)-taxifolin incubated with Aβ42 to evaluate the effects of NaIO₄ or the anaerobic condition on the autoxidation of (+)-taxifolin. When Aβ42 was incubated with (+)-taxifolin under air, the intensity of the peak at 260 and 400 nm gradually increased, and that of the peak at 320 nm decreased during 48 h of incubation (Fig. 2B). These spectral changes are characteristic of the oxidation of catechol-type flavonoids to form the o-quinone structure (44). The addition of NaIO₄ accelerated these UV changes (Fig. 2B). In contrast, there was almost no change in the UV spectra when (+)-taxifolin and Aβ42 were co-incubated in vacuo or with TCEP (Fig. 2B). These results indicate that the o-quinone formation in (+)-taxifolin through autoxidation plays a critical role in the inhibition of Aβ42 aggregation. The UV spectra of Aβ42 alone remained almost constant during the incubation (data not shown), meaning that the spectra of Aβ42 itself did not affect those of (+)-taxifolin. Conversion to the o-quinone from (±)-taxifolin in the presence of NaIO₄ was also verified by reacting with o-phenylenediamine to yield phenazine (supplemental Scheme S1B), whose structure was confirmed by ¹H NMR and high resolution EI-MS.

Effects of Autoxidation of (+)-Taxifolin on Its Ability to Inhibit Transformation of a Random Structure into a β-Sheet in Aβ42

We investigated the effects of autoxidation of (+)-taxifolin on the secondary structure of Aβ42 by using CD spectroscopy. Shown in Fig. 3A is the data for Aβ42; the positive peak at 195 nm and negative peak at 215 nm drastically increased even after 4 h of incubation, and remained until 48 h of incubation, suggesting that a random structure transformed into a β-sheet in Aβ42. On the other hand, (+)-taxifolin strongly delayed the transformation of Aβ42 (Fig. 3B). Furthermore, addition of NaIO₄ decelerated the transformation process during 0–8 h (Fig. 3C).

FIGURE 3.

Effects of autoxidation of (+)-taxifolin on its ability to prevent transformation into the β-sheet structure of Aβ42 using CD spectrometry. A-C, Aβ42 (25 μm) was incubated (A) without or with (+)-taxifolin (50 μm) in the (B) absence or (C) presence of NaIO₄ (100 μm) at 37 °C for the period indicated. D and E, Aβ42 (25 μm) was incubated (D) without or (E) with (+)-taxifolin (50 μm) in vacuo at room temperature for the period indicated.

We also measured the CD spectra under an anaerobic condition (Fig. 3, D and E). A spectrum related to β-sheet formation was found only after 24 h of incubation, but its peak intensity was weaker than that of Aβ42 under air in Fig. 3A. Because radicalization of Aβ42 induced by the reactive oxygen species is indispensable to its aggregation (8), these results seem to be reasonable. The transformation into a β-sheet was not suppressed either by (+)-taxifolin in vacuo. The findings suggest that the effects of autoxidation of (+)-taxifolin on its ability to inhibit Aβ42 aggregation are closely associated with prevention of the transformation into a β-sheet.

LC-MS Analysis of Aβ42 Treated with Oxidized Taxifolin

The o-quinone of flavonoids can form covalent bonds with nucleophilic residues in proteins (e.g. Cys, Arg, and Lys) to modulate their activity (39, 45). Because Aβ42 has three basic amino acid residues (Arg⁵, Lys¹⁶, and Lys²⁸), we asked if these residues bound to oxidized taxifolin covalently. The o-quinone derived from (+)-taxifolin can react with Lys or Arg residues in Aβ42 through a Michael addition or Schiff base formation (Fig. 4A). We analyzed an Aβ42 solution incubated with (+)-taxifolin and NaIO₄ for 4 h using a highly sensitive ion trap-type LC-MS equipped with a TOF mass analyzer (LCMS-IT-TOF). As shown in Fig. 4B, LC-MS measurements gave the mass envelop at +7, +6, and +5 charge distribution (deconvoluted mass: 4817.12, calculated: 4816.38), corresponding to the Aβ42-oxidized taxifolin adduct resulted from Michael addition. These results imply that the basic amino acid residues of Aβ42 might be involved in the covalent bonding with the oxidized taxifolin.

FIGURE 4.

LC-MS analysis of Aβ42 treated with oxidized taxifolin. A, the proposed structures of the adducts between oxidized taxifolin and Aβ42. Upper, Lys¹⁶ and Lys²⁸, and lower, Arg⁵ in Aβ42 could attack the o-quinone of (+)-taxifolin, resulting in (left) Michael addition or (right) Schiff base formation with the indicated calculated mass. B, LCMS-IT-TOF analysis of the Aβ42 solution treated with oxidized taxifolin. After Aβ42 (25 μm) was incubated with (+)-taxifolin (50 μm) in the presence of NaIO₄ (100 μm) at 37 °C for 4 h, an aliquot was subjected to the analysis.

Inhibitory Effect of (+)-Taxifolin on Aggregation of Aβ42 Mutants Substituted at Arg⁵, Lys¹⁶, and/or Lys²⁸

Although formation of Michael adducts between the o-quinone of (+)-taxifolin and the Lys residues of Aβ42 was suggested in LC-MS (Fig. 4B) together with the verification of the o-quinone formation (supplemental Scheme S1B), an attempt to determine the Lys residues involved in adduct formation by LC-MS-MS analysis was disappointing, possibly because of the extremely low amount and/or instability of the adduct. To obtain further insight into the mechanism by which (+)-taxifolin inhibits the aggregation of Aβ42, we prepared five Aβ42 mutants (R5Nle-, K16Nle-, K28Nle-, K16,K28(Nle)₂-, and R5,K16,K28(Nle)₃-Aβ42), where the basic amino acid residues of Aβ42 were substituted with Nle. The aggregative ability in the presence or absence of (+)-taxifolin was also estimated (Fig. 5, A–E). These mutants retained substantial aggregative abilities to form fibrils (70–80%) compared with wild-type Aβ42 in Th-T test (Fig. 5F). (+)-Taxifolin did not suppress the aggregative ability of K16Nle-Aβ42 (Fig. 5B). K28Nle-Aβ42 also aggregated in the presence of (+)-taxifolin, although the intensity of the Th-T fluorescence was slightly decreased than for the K28Nle-Aβ42 alone (Fig. 5C). Moreover, (+)-taxifolin did not prevent the aggregation of K16,K28(Nle)₂-Aβ42 and R5,K16,K28(Nle)₃-Aβ42 (Fig. 5, D and E). On the other hand, (+)-taxifolin largely suppressed the aggregation of R5Nlel-Aβ42 (Fig. 5A). These results indicate that lysine residues at positions 16 and 28 could be targets for the oxidized taxifolin to prevent aggregation of Aβ42. More correctly, because the aggregative ability of K28Nle-Aβ42 was slightly suppressed by (+)-taxifolin compared with that of K16Nle-Aβ42 (Fig. 5, B and C), Lys¹⁶ would be a more specific target than Lys²⁸ in inhibition of Aβ42 aggregation.

FIGURE 5.

Effects of (+)-taxifolin on the aggregation of Aβ42 mutants substituted with norleucine. The aggregative ability of: A, R5Nle-Aβ42; B, K16Nle-Aβ42; C, K28Nle-Aβ42, D, K16,K28(Nle)₂-Aβ42; and E, R5,K16,K28(Nle)₃-Aβ42 in the presence of (+)-taxifolin was examined by Th-T assay. Each Aβ42 mutant (25 μm) was incubated with or without (+)-taxifolin (50 μm) at 37 °C. The data are presented as the mean ± S.E. (n = 8). Th-T relative fluorescence was expressed as a percentage of the fluorescence for the Aβ42 mutant alone, whose maximum value was taken as 100%. *, p < 0.05 compared with Aβ42 mutant alone. The time points without asterisks means no significant difference between Aβ42 mutant treated and untreated with (+)-taxifolin. F, the comparison of aggregative ability of Aβ42 mutants. Th-T relative fluorescence of each mutant after incubation for 24 h was expressed as a percentage of the fluorescence for wild-type Aβ42 alone, whose maximum value was taken as 100%. The data are presented as the mean ± S.E. (n = 8).

Inhibition of Aβ42 Aggregation by Non-catechol-type Flavonoids

Myricetin, quercetin, morin, and kaempferol, which were previously reported to inhibit Aβ42 aggregation, belong to the flavonols (43). Flavonols contain a double bond between C2 and C3 on the C-ring, whereas flavanonols like (+)-taxifolin do not (Fig. 6A). We calculated IC₅₀ for Aβ42 aggregation from the inhibitory rate (%) of each flavonoid (10, 25, 50, and 100 μm) for a strong class inhibitor (dihydromyricetin, (+)-taxifolin, myricetin, and quercetin); 25, 50, 100, and 250 μm for a middle class inhibitor (morin, kaempferol, and datiscetin); 50, 100, 250, and 500 μm for a weak class inhibitor (dihydrokaempferol, pinobanksin, and galangin)) on the aggregation of Aβ42 (25 μm) after a 24-h incubation. The values of IC₅₀ were summarized in Fig. 6A. Among flavanonols, dihydromyricetin (IC₅₀ = 25.3 μm) as well as (+)-taxifolin (IC₅₀ = 33.0 μm) with contiguous hydroxyl groups on the B-ring suppressed the aggregation of Aβ42, whereas dihydrokaempferol and pinobanksin (IC₅₀ > 500 μm) with one or no hydroxyl group did not (Fig. 6A), suggesting vicinal hydroxyl groups on the B-ring to be essential for the inhibitory activity of flavanonols. Similarly, among flavonols, we compared the ability to inhibit Aβ42 aggregation of myricetin, quercetin, morin, kaempferol, datiscetin, and galangin. The aggregation of Aβ42 was strongly suppressed by myricetin (IC₅₀ = 15.1 μm) and quercetin (IC₅₀ = 15.3 μm) with vicinal hydroxyl groups on the B-ring, whereas galangin (IC₅₀ > 500 μm) without a hydroxyl group on the B-ring did not show any inhibitory activity (Fig. 6A). Interestingly, morin (IC₅₀ = 30.3 μm), kaempferol (IC₅₀ = 75.1 μm), and datiscetin (IC₅₀ = 55.4 μm) without a catechol moiety moderately suppressed the aggregation of Aβ42 (Fig. 6A). Regarding the relevance of autoxidation to the inhibition of Aβ42 aggregation, we measured the Th-T fluorescence of Aβ42 treated with these three non-catechol-type flavonols under an anaerobic condition or in the presence of TCEP. These flavonols suppressed the aggregation of Aβ42 even in vacuo (Fig. 6B). Moreover, addition of excess TCEP (Aβ42:flavonols:TCEP = 25:50:200 μm) did not affect the suppressive ability of these flavonols (data not shown), indicating that the inhibition of non-catechol-type flavonols could not be ascribed to their autoxidation.

FIGURE 6.

Effects of flavanonols and flavonols on the aggregation of Aβ42. A, the structures and IC₅₀ values of (upper) flavanonols and (lower) flavonols examined in this study. The IC₅₀ value was calculated from the inhibitory rate (%) of each flavonoid on Aβ42 aggregation after a 24-h incubation using Th-T assay. B, Aβ42 (25 μm) incubated with morin, kaempferol, or datiscetin (50 μm) in vacuo at room temperature. Th-T relative fluorescence was expressed as a percentage of the fluorescence for Aβ42 alone, whose maximum value was taken as 100%.

To gain further insight into the mechanism by which flavonoids inhibit Aβ42 aggregation by targeting the Lys residues, aggregation tests were carried out in the presence of catechol-type flavonoids (dihydromyricetin, (+)-taxifolin, myricetin, or quercetin), or non-catechol-type flavonols (morin, kaempferol, or datiscetin) using K16Nle- and K16,K28(Nle)₂-Aβ42. We compared the aggregative ability of the Aβ42 mutant (25 μm) incubated with each flavonoid (50 μm), the concentration of which was the maximal value to suppress the fluorescence of Aβ42 under 50% by (+)-taxifolin (data not shown). The catechol-type (+)-taxifolin and quercetin did not suppress the aggregation of these Aβ42 mutants. Although dihydromyricetin and myricetin with contiguous trihydroxyl groups significantly prevented the aggregation of K16Nle-Aβ42, these flavonoids did not change the aggregative potency of K16,K28(Nle)₂-Aβ42 (Fig. 7A), implying that they could react with Lys²⁸ as well as Lys¹⁶ because contiguous trihydroxyl groups might facilitate the autoxidation of the B-ring compared with (+)-taxifolin and quercetin containing vicinal hydroxyl groups. Notably, in the case of non-catechol-type flavonols (morin, kaempferol, and datiscetin), there was little difference in the inhibitory activity among the wild-type, K16Nle-Aβ42, and K16,K28(Nle)₂-Aβ42 (Fig. 7B). These results suggest the existence of another inhibitory mechanism for Aβ42 aggregation by non-catechol-type flavonols other than the autoxidation followed by the Michael addition of Lys residues, as observed for (+)-taxifolin.

FIGURE 7.

Effects of flavanonols and flavonols on the aggregation of Aβ42 mutants. A, the aggregative ability of Aβ42 mutants (25 μm) incubated with a catechol-type flavanonol or flavonol (50 μm) for 24 h estimated by Th-T tests. The data are presented as the mean ± S.E. (n = 8). Th-T relative fluorescence was expressed as a percentage of the fluorescence for each Aβ42 alone, whose value at 24 h was taken as 100%. B, the aggregative ability of Aβ42 mutants (25 μm) treated with a non-catechol-type flavonol (50 μm) for 24 h, estimated by Th-T tests. *, p < 0.05. n.s., not significant.

Analysis of the Interaction of Aβ42 Aggregates with (+)-Taxifolin Using Solid-state NMR

Our recent study using a solid-state NMR showed that curcumin with an α,β-unsaturated ketone interacted with the aromatic hydrophobic core (Aβ17–21) due to its inherent hydrophobicity and planarity, resulting in the inhibition of Aβ42 aggregation via intercalation (18). Curcumin was reported to interact with Aβ40 fibrils through the planarity of the enol form of curcumin (46). Also given are the preferable detection of monomeric Aβ42 in LC-MS analysis, similar analysis was performed to clarify the interaction between Aβ42 and (+)-taxifolin. (+)-Taxifolin was labeled with ¹³C₆ on the B-ring based on previous structure-activity relationship studies, in which the catechol moiety on the B-ring is critical in the inhibitory potential, and the methylation of the hydroxyl group at position 7 on the A-ring did not influence the aggregation of Aβ42 (23). Aβ42 was also ¹³C-labeled site-specifically at Ala², Ser⁸, Lys¹⁶, Val¹⁸, Phe¹⁹, and Phe²⁰, in which only C_β was labeled in Phe¹⁹ and Phe²⁰ to avoid overlapping the Aβ42 and (+)-taxifolin signals. For the broad band ¹³C-¹³C correlation two-dimensional experiments, dipolar-assisted rotational resonance was employed (34). As shown in supplemental Fig. S2B, the interaction peaks between Aβ42 and (+)-taxifolin were as weak as noise signals despite the use of a 10-fold excess of (+)-taxifolin (Aβ42:(+)-taxifolin = 13:145 μm), whereas a 5-fold excess was employed for curcumin (Aβ42:curcumin = 10:50 μm) (18). Remarkably, the ¹³C-¹³C cross-peaks between Lys residues and the B-ring of (+)-taxifolin was not observed significantly (supplemental Fig. S2B). More specifically, the interaction of (+)-taxifolin with the aromatic hydrophobic core (Aβ17–21), which was previously found in the curcumin case due to its inherent hydrophobicity and planarity (18) was not observed. These indicate that the inhibitory mechanism of Aβ42 aggregation by (+)-taxifolin (via covalent bonding) could be different from that of curcumin (via intercalation).

Because lack of the double bond at positions 2 and 3 of (+)-taxifolin could decrease its planarity, it might not be able to insert into the β-sheet region of the Aβ42 aggregate (Lys¹⁶–Ala²¹). Instability of the oxidized taxifolin-Aβ42 adduct might be another reason. The previous solution-state NMR studies on myricetin and Aβ42 (20) suggested the less specific broad recognition of the β-sheet region, possibly due to the usage of excessive amounts of myricetin (Aβ42:myricetin = 25:200 μm). This finding might reflect the difference in suppressive ability between myricetin and (+)-taxifolin against K16Nle-Aβ42 (Fig. 7A).

DISCUSSION

Thus far, the anti-AD activity of flavonoids has been believed to originate from their antioxidative activity and/or β-sheet recognition due to their hydrophobicity and planarity. However, these parameters are not necessarily accompanied by the ability to inhibit the aggregation of Aβ42 and other amyloidogenic proteins (11). This background led us to reconsider whether the inhibitory activity can be simply explained by these “less specific” properties (antioxidation, hydrophobicity, and planarity) or not.

On the basis of the present results, we propose a site-specific mechanism whereby catechol-type flavonoids inhibit the aggregation of Aβ42, in which a catechol structure could be autoxidized to form the o-quinone on the B-ring, followed by the formation of the o-quinone-Aβ42 adduct targeting Lys residues at positions 16 and 28 of Aβ42, but not be originated from the antioxidative activity (Fig. 8). This could provide unique opportunities to design potent inhibitors of Aβ42 aggregation. On the other hand, the inhibitory ability of non-catechol-type flavonols containing a double bond between C2 and C3 on the C-ring (Fig. 6A) does not require the autoxidation. The data in Figs. 6 and 7 indicate that the interaction of non-catechol-type flavonols with Aβ42 might be less effective than the conjugate addition of the Lys residues to the o-quinone moiety derived from autoxidation. These findings might explain in part the difference in the inhibitory ability between flavanonols and flavonols.

FIGURE 8.

Site-specific inhibitory mechanisms of Aβ42 aggregation by catechol-type flavonoids. Catechol-type flavanonols (e.g. (+)-taxifolin) or flavonols (e.g. quercetin) were oxidized to form corresponding o-quinones on B-ring, followed by the formation of adducts by Lys¹⁶ and Lys²⁸ of Aβ42. Because Lys¹⁶ and Lys²⁸ are incorporated in the intermolecular β-sheet region (8), Aβ42 aggregates would be destabilized by the adduct formation.

Our previous investigation using solid-state NMR together with systematic proline replacement identified a toxic conformer with a turn at positions 22 and 23 in Aβ42 (47), and proposed that the residues at positions 15–21 and 24–32 containing Lys¹⁶ and Lys²⁸ are involved in the intermolecular β-sheet region, whereas the N-terminal 13 residues are not (48). A monoclonal antibody against the toxic turn at positions 22 and 23 detected Aβ oligomers in human AD brain (49) and induced pluripotent stem cells (50) as well as in AD mice (51, 52). As mentioned above, an attempt to determine the Lys residues involved in adduct formation by LC-MS-MS analysis gave disappointing results, possibly because of the extremely low amount and/or instability of the adducts. Because the targeted Lys residues (Lys¹⁶ and Lys²⁸), not the Arg residue (Arg⁵), are incorporated in the intermolecular β-sheet region (Fig. 8), even a small amount of covalently bonded adducts at the Lys residues of Aβ42 oligomers and/or protofibrils could inhibit the formation of Aβ42 aggregates (fibrils) detected by the Th-T fluorescence.

Recently, Bitan's group reported that the Lys-specific synthetic compound (molecular tweezer, CLR01) prevented cytotoxicity and oligomerization of Aβ42 through non-covalent interaction in vitro (53) and in vivo (54). Their subsequent study using Aβ42 mutants substituted at Lys¹⁶ or Lys²⁸ with Ala revealed a key role for Lys residues in Aβ42-induced neurotoxicity rather than aggregation (55). These reports did not contradict the results that the aggregates of double and triple mutants (K16,K28(Nle)₂-Aβ42 and R5,K16,K28(Nle)₃-Aβ42) after 24 h of incubation were slightly less than that of wild-type Aβ42 in this study (Fig. 5F).

R5Nle-Aβ42 (∼4 h) and K28Nle-Aβ42 (∼2 h) had the nucleation phase (Fig. 5, A and C), whereas three Aβ42 mutants (K16Nle-Aβ42, K16,K28(Nle)₂-Aβ42, and R5,K16,K28(Nle)₃-Aβ42) including the substitution of Lys¹⁶ with Nle did not (Fig. 5, B, D, and E). These mean that the aggregative ability of K16Nle-Aβ42 seems to be more potent than that of K28Nle-Aβ42. Because Lys¹⁶ is located at a hydrophobic cavity in the β-sheet region (56), the substitution with norleucine without an amino group could enhance the hydrophobic interaction in Aβ42 aggregates, leading to passing the nucleation phase. In contrast, Lys²⁸ was involved in the formation of the salt bridge between Asp²³ and Lys²⁸ for Aβ42 aggregation (56). These findings imply the different role of lysine resides at positions 16 and 28 in Aβ42 aggregation, which might explain the difference of aggregative ability between K16Nle- and K28Nle-Aβ42.

Notably, the nucleation phase of R5Nle-Aβ42 (∼4 h) was partially longer than that of wild-type Aβ42 (∼1 h) (Fig. 1, A, B, and F). Because flexibility of the N-terminal region has been thought to be essential for aggregation of Aβ42 (56), the replacement of Arg⁵ with norleucine might retard the nucleation phase in wild-type Aβ42 aggregation by increasing the hydrophobic interaction. Moreover, given no nucleation phase of the three Aβ42 mutants of Lys¹⁶, it is not surprising that (+)-taxifolin did not largely alter the aggregation properties of these mutants, because (+)-taxifolin could specifically target the elongation phase in wild-type Aβ42 aggregation, rather than the nucleation phase.

LeVine et al. (57) suggested preventive effects on Aβ42 aggregation by several dihydroxybenzoic acid isomers, in which 2,3- and 3,4-dihydroxy benzoic acids delayed the velocity of oligomer formation. Fisetin, a quercetin analog without the 5-OH group on the A-ring, also inhibited the aggregation of Aβ42 (58). Ushikubo et al. (59) reported that the 7-OH group on the A-ring is not involved in the anti-aggregative ability of flavonols. These are consistent with our previous study on the structure-activity relationship of (+)-taxifolin (23). On the other hand, lacmoid without a catechol moiety bound less specifically to Aβ42 (60). These findings together with the present results strongly support that flavonoids with vicinal hydroxyl groups on the B-ring could be indispensable to bind covalently with Aβ42 to suppress its aggregation. It is also reasonable that 3,4,5-trihydroxybenzoic acid, gallic acid (57), and 3′,4′,5′-trihydroxyflavone (59) as well as dihydromyricetin (Fig. 6A) suppressed the aggregation of Aβ42. In particular, the inhibitory activities of dihydromyricetin, (+)-taxifolin, myricetin, and quercetin were higher than those of morin, kampferol, and datiscetin (Fig. 6A), suggesting that nucleophilic addition to the o-quinone moiety by the Lys residues of Aβ42 could contribute more significantly to the inhibition of Aβ42 aggregation than the hydrophobic interaction. However, an additional role of the A- and C-rings of catechol-type flavonoids in the suppression of Aβ42 aggregation is not negligible, because the inhibitory activity of catechol itself was low (61).

Fink and colleagues (45) previously proposed the contribution of interaction of Lys residues with the baicalein o-quinone to the inhibition of α-synuclein responsible for Parkinson disease. However, the underlying molecular mechanism cannot be fully explained by the oxidized baicalein because the aggregation of α-synuclein was inhibited by baicalein even under an anaerobic condition (45). Quite recently, the oxidation product of EGCG was in part involved in remodeling the preformed fibrils of Aβ40 by EGCG (62).

To the best of our knowledge, this is a first report that dihydromyricetin and datiscetin as well as (+)-taxifolin have anti-aggregative activity against Aβ42. We also demonstrated that (+)-taxifolin could suppress the elongation phase in the aggregation of wild-type Aβ42, rather than the nucleation phase. Seed extracts of S. marianum, known as silymarin, have long been used as an anti-hepatotoxic medicine without notable adverse effects (63), and in particular, are efficacious against damage induced by alcohol and disturbances in the function of the gastrointestinal tract (64). Booth and Deeds (65) showed that (+)-taxifolin was not toxic when given long term to albino rats. Therefore, (+)-taxifolin may be a worthy candidate for AD therapeutics. Although some polyphenols (naringenin (66) and curcumin (67)) were reported to pass through the blood-brain barrier after oral administration, caution should be used because of the differences between animal and clinical conditions.