Effect of natural antioxidants on the aggregation and disaggregation of beta-amyloid

Purpose: To examine the relationship between higher antioxidant activity and aggregation or disaggregation of beta-amyloid (Aβ) for 21 plants. Methods: Twenty-nine natural plant extracts and their antioxidant activities were analyzed using DPPH assay. The aggregation and disaggregation of Aβ were analyzed using Thioflavin-T assay. Results: Eleven plant extracts exhibited high antioxidant activities with the half-maximal inhibitory concentration (IC50) values < 20.0 μg/mL. Furthermore, the plant extracts efficiently inhibited Aβ aggregation with a mean IC50 value of 17.0 μg/mL. However, four plant extracts exhibiting low antioxidant activities (IC50 > 80.0 μg/mL) inhibited Aβ aggregation less efficiently with a mean IC50 value of 75.7 μg/mL. Furthermore, plant extracts with high antioxidant activities were not invariably efficient for disaggregating pre-formed Aβ aggregates. Conclusion: High antioxidant activities were positively correlated with the inhibition of Aβ aggregation, although not with the disaggregation of pre-formed Aβ aggregates. Nevertheless, potent antioxidants may be helpful in treating Alzheimer’s disease.


INTRODUCTION
β-amyloid (Aβ) is a polypeptide of 36-43 amino acids, and is a major causative factor of Alzheimer's disease (AD).Aβ aggregation into senile plaques is a pathological characteristic detected in the brains of patients with AD.Aβ is produced by the sequential cleavage of amyloid precursor proteins by β-and γ-secretases.Aβ molecules undergo self-aggregation to form oligomers and fibrils, which cause neurotoxicity via oxidative stress, disturb calcium homeostasis, and induce mitochondrial dysfunction, and neuroinflammation.Thus, the discovery of compounds or extracts that inhibit Aβ aggregation, and induce the disaggregation of Aβ aggregates could be an effective approach to the therapy and prevention of AD.
Antioxidants are molecules that scavenge free radicals and reactive oxygen species (ROS), which can induce oxidative stress, and consequently, cellular damages.Antioxidant intake is helpful in preventing human diseases [1].For example, antioxidants can reduce the risk of cardiovascular diseases by inhibiting vascular inflammation, lipid peroxidation, and platelet aggregation [2].Additionally, antioxidants are known to have anti-cancer, anti-diabetic, and anti-aging effects [3][4][5].Antioxidants are also considered helpful in treating AD, because the ROS generated in AD pathology causes neuronal damage [6].Furthermore, certain antioxidants such as polyphenols inhibit Aβ aggregation, and destabilize preformed Aβ aggregates [7,8].However, the relationships between antioxidant activity and inhibition of Aβ aggregation, and between antioxidant activity and disaggregation of preformed Aβ aggregates are not analyzed.Therefore, in this study, 29 natural plant extracts with diverse antioxidant activity were analyzed for effects on the aggregation and disaggregation of Aβ, and the correlation between them was analyzed as well.

Plant materials
Twenty-nine plant materials (Table 1) were purchased from a commercial market (Samhing medicinal herb market; Seoul, Korea) in 2014.One of the authors (S-Y Park) performed botanical identification and voucher specimens were deposited in Pharmacognosy laboratory of College of Pharmacy, Dankook University.

Preparation of plant extracts
Dried and pulverized plant materials were extracted 3 times with 90 % methanol at room temperature.The MeOH filtrate was evaporated under vacuum to yield the MeOH extract.Each extract was dissolved in DMSO and stored at -20 °C until use for the assay.

Antioxidant assay
The antioxidant activities of the plant extracts were determined using a stable free radical, DPPH.The ability of the plant extracts to scavenge DPPH, and convert it to 1,1-diphenyl-2-picrylhydrazine was determined colorimetrically.In brief, the plant extracts at different concentrations of (4, 20, and 100 μg/mL) were mixed with 190 μL of DPPH (0.316 mM in ethanol) and the mixtures were incubated at 37 °C for 30 min.The optical density values of the mixtures were measured at 517 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).All measurements were conducted out at least thrice.

ThT assay of inhibition of Aβ aggregation
To quantify Aβ aggregates, the ThT assay was performed.Aβ 1-42 was dissolved in DMSO at 1 mL, and stored at -20 °C until use.To monitor their effects on Aβ aggregation, the plant extracts (4, 20, and 100 µg/mL) were incubated with 20 µM of Aβ (49 µL) at 37 °C for 24 h.Then, 3 µM of ThT (50 µL) was added, and the fluorescence was measured after 30 min with an EMax precision microplate reader (Molecular Devices) with excitation at 442 nm and emission at 485 nm.Aβ treated with DMSO was used as the control, and each assay was performed in triplicate.

ThT assay of disaggregation of preformed Aβ aggregates
To monitor the effects of the plant extracts on the disaggregation of the Aβ aggregates, 20 µM of Aβ (49 µL) was incubated at 37 °C.After 24 h, the plant extracts (4, 20, and 100 µg/mL) were added, and incubated for an additional 24 h.Subsequently, 3 µM of ThT (50 µL) was added, and the fluorescence was measured as described in the previous section.

Statistical analysis
Data were expressed as means ± SD.Two or more group comparisons were performed by one-way analysis of variance (ANOVA), followed by Tukey's post-hoc test (SPSS version 17.0; IBM SPSS, Armonk, NY, USA).Differences were considered statistically significant at P < 0.05.

Inhibition of Aβ aggregation by the plant extracts
The ThT assay demonstrated that Cornus officinalis exhibited the highest inhibitory effect on Aβ aggregation with an IC 50 value of 8.2 μg/mL (Table 1).1B).

Effects of the plant extracts on disaggregation of Aβ aggregates
The disaggregating effects of the 29 plant extracts on pre-aggregated Aβ aggregates were determined by the ThT assay.Thuja orientalis exhibited the highest disaggregating activity among the 29 plant extracts with an IC 50 value of 14.7 μg/mL (Table 1).Similarly, Leonurus japonicus efficiently induced the disaggregation of preformed Aβ aggregates with an IC 50 value of 25.7 μg/mL.Contrarily, the effects of Torilis japonica and Rehmannia glutinosa on the disaggregation of preformed Aβ aggregates were extremely minimal with IC 50 values of 377.6 and 396.9 μg/mL, respectively.

Antioxidant activity and inhibition of Aβ aggregation
To determine the relationship between the inhibition of Aβ aggregation and antioxidant  and 4.These results indicated that the plant extracts with higher antioxidant activities exhibited higher anti-Aβ aggregating activities.Therefore, plants with higher antioxidant activities could be beneficial for patients with AD because of their possibly higher anti-Aβ aggregating activities.

Antioxidant activity and disaggregation of Aβ aggregates
To determine the relationship between antioxidant activity and the disaggregation of Aβ aggregates, the mean IC 50 values of groups 1-4 for the disaggregation of Aβ aggregates were compared (Figure 2C).The average IC 50 values of the groups 1, 2, 3, and 4 for Aβ disaggregation were 84.3, 107.6, 105.8, and 291.2 μg/mL, respectively.Group 1 exhibited a relatively low IC 50 value for Aβ disaggregation; however, it was not significantly different from those of groups 2 and 3.However, the disaggregating activities of groups 1, 2, and 3 were significantly different from that of group 4.

DISCUSSION
Natural products containing polyphenolic compounds, such as berries, spices, and green tea, are known to have antioxidant and neuroprotective activities.Furthermore, the neuroprotective effects of natural polyphenolic compounds, such as myricetin and luteolin are exerted, at least partially, by attenuating Aβinduced toxicity [9].The aggregation of Aβ monomers to form oligomers and fibrils (aggregates) induces neurotoxicity.Oxidative stress, induced during Aβ aggregation, disrupts synapses, and impairs the functions of synapses and membranes [10,11].Thus, antioxidants could be helpful in treating AD by reducing oxidative stress-related neuronal damage.Additionally, the use of compounds or extracts inhibiting Aβ aggregation is considered a good approach to the therapy and prevention of AD.For example, flavonoids such as myricetin, quercetin, and kaempferol inhibited Aβ aggregation (exhibited antiamyloidogenic activities) [12][13][14].Additionally, rosmarinic acid and curcumin are other compounds that inhibit Aβ aggregation efficiently [15,16].Interestingly, these compounds are known to have high antioxidant activities.Consistent with previous reports, our results suggested that the plant extracts with relatively high antioxidant activity inhibited Aβ aggregation efficiently.This could be because potent antioxidants, such as polyphenols, in the plant extracts interact with aromatic regions in Aβ, disrupt the self-assembly of Aβ into β-sheet conformational stacking, and eventually, inhibit Aβ aggregation [17].
Antioxidants are also considered efficient in disaggregating Aβ.Curcumin and β-carotene exhibited high Aβ-disaggregating activities owing to their symmetric rod-like structures and hydrophobic moieties that wedged into Aβ cores, and disrupt the β-sheet structures [18,19].However, the increases in hydrophilicity reduced Aβ-disaggregating activity [18].Furthermore, morin is an efficient Aβ-disaggregating flavonoid.The aromatic moiety and hydrogen-bonding capacity of morin are suggested to cause the disaggregation of Aβ aggregates [20].These reports suggest that molecules with hydrophobicity, aromatic moiety, and hydrogen bonding-capacity might, at least, partially contribute to the disaggregation of Aβ aggregates.Consistent with these results, our study similarly suggested that antioxidants are not invariably positively correlated with high Aβdisaggregating activity.Altogether, these results suggested that the plant extracts with high antioxidant activities are efficient in inhibiting Aβ aggregation; however, they were not invariably efficient in disaggregating Aβ aggregates.Nonetheless, antioxidants could be beneficial for patients with AD owing to their possible anti-Aβ aggregating and Aβ-disaggregating activities.

CONCLUSION
Twenty-nine plant extracts have been analyzed for antioxidant, anti-amyloidogenic, and Aβ disaggregating activities.The plant extracts with high antioxidant activity inhibit Aβ aggregation more efficiently than those with low antioxidant activity; however, antioxidant activity correlates weakly with the disaggregation of pre-formed Aβ aggregates.Nevertheless, good antioxidants may be helpful in treating AD.

Figure 2 :
Figure 2: Mean values of the antioxidant, anti-Aβ aggregating and Aβ-disaggregating activities of 29 plant extracts divided into 4 groups based on antioxidant activity.(A) Average values of antioxidant activities, (B) average values of anti-Aβ aggregating activities and (C) average values of Aβ-disaggregating activities.a P < 0.05, different from Group 1, b P < 0.05, different from Group 2, c P < 0.05, different from Group 3, d P < 0.05, different from Group 4 However, this does not indicate that potent antioxidants are efficient in the inhibition of Aβ aggregation and disaggregation of Aβ aggregates.Therefore, in this study, the relationships between antioxidant activity and inhibition of Aβ aggregation, and between antioxidant activity and disaggregation of preformed Aβ aggregates were examined.