Catechol-Type Flavonoids from the Branches of Elaeagnus glabra f. oxyphylla Exert Antioxidant Activity and an Inhibitory Effect on Amyloid-β Aggregation

Elaeagnus glabra f. oxyphylla (Elaeagnaceae) is a small evergreen tree with narrow lanceolate leaves that is native to Korea. In this work, we studied the chemical composition of E. glabra f. oxyphylla branches (EGFOB) for the first time. Additionally, we evaluated the effects of the ethanol extract of EGFOB and each of its chemical components on key mediators of Alzheimer’s disease (AD), namely, amyloid-β (Aβ) aggregation and oxidative stress. The ethanol extract of EGFOB decreased Aβ aggregation (IC50 = 32.01 µg/mL) and the levels of the oxidative free radicals 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (IC50 = 11.35 and 12.32 µg/mL, respectively). Sixteen compounds were isolated from EGFOB. Among them, procyanidin B3 (8), procyanidin B4 (9), and helichrysoside (13) significantly inhibited Aβ aggregation (IC50 = 14.59, 32.64, and 44.45 μM, respectively), indicating their potential as bioactive compounds to control Aβ aggregation. Furthermore, these compounds markedly enhanced in vitro scavenging activity against ABTS (IC50 = 3.21–4.61 µM). In the DPPH test, they showed lower scavenging activity than in the ABTS test (IC50 ≥ 54.88 µM). Thus, these results suggest that EGFOB and specifically compounds 8, 9, and 13 may be beneficial in AD prevention and treatment through their antioxidant and anti-Aβ aggregation activities.


Introduction
Alzheimer's disease (AD) is a progressive neurodegenerative disorder with no available cure. The incidence rate is higher among people over the age of 60, with AD patients accounting for 60-80% of all dementia cases [1,2]. Currently, the number of dementia patients in the world is estimated at 44 million, and this number is predicted to more than triple by 2050 [3].
AD is characterized by cognitive disruptions, such as memory loss and language difficulties, and non-cognitive dysfunctions, such as behavioral disturbance, depression, hallucination, and delusion [4,5]. The hallmark of AD pathogenesis is the progressive accumulation of amyloid-β (Aβ) in the brain [6]. Aβ plaques are extracellular accumulations of pathological forms of Aβ that are principally composed of abnormally folded Aβ, such as Aβ  and Aβ  . Because of its higher rates of fibrillation and insolubility, Aβ 1-42 is more abundant in the plaques than Aβ 1-40 [3]. Aβ aggregation can be initiated by oxidative stress in the early stage of AD, and Aβ plaques also influence free radical

Inhibitory Effects of EGFOB and EGFOL on Aβ Aggregation
To explore whether E. glabra f. oxyphylla has an inhibitory effect on Aβ aggregation, we prepared ethanol extracts of EGFOB and EGFOL at various concentrations (6.25, 12.5, 25, 50, or 100 µg/mL). Both EGFOB and EGFOL extracts inhibited in vitro Aβ aggregation in a dose-dependent manner (IC 50 = 32.01 and 92.97 µg/mL, respectively) ( Figure 1A, B). These results indicate that EGFOB have a stronger inhibitory effect on Aβ aggregation compared with EGFOL. Aβ aggregation is one of the primary pathological indicators in AD patients [6]. Despite continuous debate about the relevance of Aβ as a target biomarker in new drug development for AD, many researchers are still focusing on Aβ [25][26][27]. The amyloid hypothesis suggests that the abnormality of Aβ is observed somewhat earlier before the onset of AD compared with other AD biomarkers [28]. Therefore, Aβ should be considered an important priority in investigations for the prevention as well as the treatment of AD. In our study, the robust activity of EGFOB is a notable result for anti-Aβ therapy research.  (6.25, 12.5, 25, 50, or 100 µg/mL) of (A) EGFOB and (B) EGFOL were prepared and reacted with Aβ 1-42 peptides, followed by the addition of Thioflavin (ThT). Fluorescence intensity was measured at 440 nm (excitation) and 485 nm (emission). Each value is expressed as the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. control.

Antioxidant Effects of EGFOB and EGFOL
Oxidative stress induced by Aβ in the brain is related to the pathogenesis of AD [11]. Previous studies have reported that many natural products have robust antioxidant properties [29,30]. To examine the antioxidant activities of EGFOB and EGFOL, 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays, widely used to assess the antioxidant efficacy of natural products [29,30], were performed. The EGFOB and EGFOL extracts reached 100% ABTS radical scavenging activity at the concentrations of 25 and 100 µg/mL, respectively (IC 50 = 11.35 and 26.01 µg/mL, respectively) (Figure 2A,B). While the EGFOB extract showed similar results in the DPPH assay to those in the ABTS assay, the EGFOL extract had a significant effect on DPPH radical scavenging only at 100 µg/mL (IC 50 = 12.32 and >100 µg/mL, respectively) ( Figure 2C,D). The remarkable radical scavenging activity of EGFOB extract against ABTS and DPPH indicates the antioxidant capacity of EGFOB. Previous studies have been reported focusing on the relationship between free radicals and the formation of Aβ aggregates in AD development. Aβ plaques contributes to the generation of free radicals and oxidative stress [31], and it was found that oxidative stress is closely related to Aβ aggregation [32]. As is well known, there are many natural products that are powerful antioxidants and can play an important role in the regulation of various chronic diseases, including AD. In new drug development for AD, natural extracts such as EGFOB are attractive agents with dual-or multi-function. By comparing the effects of the two extracts in the Aβ aggregation and radical scavenging assays according to the IC 50 values, it was found that the EGFOB extract was more effective than the EGFOL extract.

Inhibitory Effects of the Solvent Fractions of EGFOB on Aβ Aggregation
The ethanol extract of EGFOB was suspended in water and successively fractionated by using n-hexane, ethyl acetate (EtOAc), and n-butanol (n-BuOH) ( Figure 3). The Aβ aggregation assay was performed using the water, n-hexane, EtOAc, and n-BuOH fractions. As shown in Figure 4, the EtOAcand n-BuOH-soluble fractions significantly inhibited Aβ aggregation (IC 50 = 24.31 and 30.24 µg/mL, respectively), unlike the other fractions. This observation indicates that the EtOAc-and n-BuOH-soluble fractions contain the bioactive compounds of EGFOB against Aβ aggregation.   water fractions were prepared and reacted with Aβ 1-42 peptides, followed by the addition of ThT. Fluorescence intensity was measured at 440 nm (excitation) and 485 nm (emission). Each value is expressed as the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. control.

Inhibitory Effects of Compounds 1-16 from EGFOB on Aβ Aggregation
We evaluated the biological activities of the sixteen compounds through an in vitro Aβ aggregation assay and a free radical scavenging assay. Compounds 8, 9, and 13 inhibited Aβ aggregation comparably to morin, a known inhibitor of Aβ aggregation [49] ( Figure 7A). Subsequently, we evaluated the inhibitory effects of these compounds on Aβ aggregation at various concentrations (6.25, 12.5, 25, 50, or 100 µM). Compounds 8, 9, and 13 significantly inhibited Aβ aggregation in a dose-dependent manner (IC 50 = 14.59, 32.64, and 44.45 µM, respectively) ( Figure 7B-D). These results indicate the potential of compounds 8, 9, and 13 as bioactive compounds to control Aβ aggregation. The inhibitory rates of the other thirteen compounds were <63%.

Antioxidant Effects of Compounds 1-16 from EGFOB
To examine the antioxidant activities of the sixteen compounds isolated from EGFOB, ABTS and DPPH radical scavenging assays were performed. Twelve compounds, excluding compounds 1, 2, 14, and 15, significantly increased radical scavenging activity against ABTS at 100 µM ( Figure 8A). The scavenging activities of compounds 1, 2, 14, and 15 against ABTS were <66%. However, the DPPH radical scavenging activities of the sixteen compounds were <56% ( Figure 8B). Additionally, we evaluated the ABTS and DPPH radical scavenging activities of compounds 8, 9, and 13, candidate inhibitors of Aβ aggregation, at various concentrations from 1.5625 to 100 µM. As shown in Figure 8C-E, compounds 8 (IC 50 = 3.21 µM), 9 (IC 50 = 3.44 µM), and 13 (IC 50 = 4.61 µM) dose-dependently enhanced radical scavenging activity against ABTS, which reached 100% at 12.5, 12.5, and 25 µM, respectively. In the DPPH test, we observed that the scavenging activity patterns of compounds 8, 9, and 13 were similar to those detected in the ABTS test. However, the DPPH radical scavenging activities of these three compounds were <52% (IC 50 = 54.88, >100, and >100 µM, respectively). As mentioned above, since Aβ aggregation and free radicals are closely related, compounds 8, 9, and 13 targeting Aβ with potent antioxidant activity are promising as valuable candidates for the prevention and treatment of AD. Additional studies will be necessary to identify the bioactive compounds of EGFOB in the inhibition of AD pathogenesis. In vitro and in vivo assays are required to determine the efficacy and safety of each candidate bioactive compound.  5625-100 µM) were measured using ABTS and DPPH radical scavenging assays. Ascorbic acid (AA) (50 µM) was used as a positive control. Each value is presented as the mean ± SEM (n = 3). * p < 0.05, *** p < 0.001, or **** p < 0.0001 vs. control.

Plant Materials
Dried EGFOB and ethanol extracts of EGFOL were obtained from the Korean Seed Association. The origins of the herbal materials were identified by Professor Joo-Hwan Kim (Gachon University, Seongnam, Korea). A voucher specimen (SCD-A-112) was deposited at the Clinical Medicine Division of Korea Institute of Oriental Medicine (Daejeon, Korea).

Aβ Aggregation Assay
To measure the inhibitory effect on Aβ 1-42 aggregation, the SensoLyte ® Thioflavin T (ThT) β-Amyloid aggregation kit (AnaSpec, Fremont, CA, USA) was used according to the modified manufacturer's instructions. This assay is based on the property of ThT dye that increases fluorescence when bound to the aggregates of Aβ 1-42 peptides. The detailed protocol is described in a previous report [53]. Morin (100 µM) was used as a positive control for the inhibition of Aβ aggregation. Experiments were performed in triplicate and independently repeated three times. The inhibition rate (%) of Aβ aggregation was calculated according to the following equation: Inhibition of Aβ aggregation (%) = 1 − Fluorescence of Aβ − treated sample Fluorescence of untreated sample × 100 (1)

Free Radical Scavenging Assay
ABTS and DPPH radical scavenging assays were performed with reference to the modified method described in previously published reports [54,55] to examine the antioxidant activities. In the ABTS assay, 7 mM ABTS aqueous solution and 2.45 mM potassium persulfate were reacted for 12-16 h in the dark at room temperature to prepare the ABTS •+ solution. Subsequently, the reactant was diluted 25-fold to adjust its absorbance at 734 nm to 0.7 by using a spectrophotometer (Benchmark Plus, Bio-Rad, Hercules, CA). The sample solution (100 µL) and ABTS •+ solution (100 µL) were mixed and incubated for 5 min at room temperature in the dark.
To determine the DPPH radical scavenging activity, the sample solution (100 µL) and 0.15 mM DPPH solution (100 µL) were mixed and incubated for 3 h at room temperature in the dark.
The absorbance was measured at 517 nm. Ascorbic acid was used as a positive control for antioxidative activity. The radical scavenging activity (%) was calculated using the following equation: Radical scavenging activity (%) = 1 − Absorbance of sample Absorbance of control × 100 (2)

Statistical Analysis
Calculated values were expressed as mean ± SEM. One-way analysis of variance or Student's t-test was performed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA) to determine statistical significance. P-values <0.05 were considered statistically significant.