Neuroprotective Effects of Davallia mariesii Roots and Its Active Constituents on Scopolamine-Induced Memory Impairment in In Vivo and In Vitro Studies

Abstract

Beta-amyloid (Aβ) proteins, major contributors to Alzheimer's disease (AD), are overproduced and accumulate as oligomers and fibrils. These protein accumulations lead to significant changes in neuronal structure and function, ultimately resulting in the neuronal cell death observed in AD. Consequently, substances that can inhibit Aβ production and/or accumulation are of great interest for AD prevention and treatment. In the course of an ongoing search for natural products, the roots of Davallia mariesii T. Moore ex Baker were selected as a promising candidate with anti-amyloidogenic effects. The ethanol extract of D. mariesii roots, along with its active constituents, not only markedly reduced Aβ production by decreasing β-secretase expression in APP-CHO cells (Chinese hamster ovary cells which stably express amyloid precursor proteins), but also exhibited the ability to diminish Aβ aggregation while enhancing the disaggregation of Aβ aggregates, as determined through the Thioflavin T (Th T) assay. Furthermore, in an in vivo study, the extract of D. mariesii roots showed potential (a tendency) for mitigating scopolamine-induced memory impairment, as evidenced by results from the Morris water maze test and the passive avoidance test, which correlated with reduced Aβ deposition. Additionally, the levels of acetylcholine were significantly elevated, and acetylcholinesterase levels significantly decreased in the brains of mice (whole brains). The treatment with the extract of D. mariesii roots also led to upregulated brain-derived neurotrophic factor (BDNF) and phospho-cAMP response element-binding protein (p-CREB) in the hippocampal region. These findings suggest that the extract of D. mariesii roots, along with its active constituents, may offer neuroprotective effects against AD. Consequently, there is potential for the development of the extract of D. mariesii roots and its active constituents as effective therapeutic or preventative agents for AD.

Keywords: Aβ aggregation; Aβ production; ethanol extract of D. mariesii roots; flavonoids; scopolamine-induced memory impairment.

Figure 1

The effect of DME on sAPPβ and β-secretase. (A) The levels of sAPPβ and β-secretase in APP–CHO cells treated with various concentrations (100, 50, 25, and 12.5 μg/mL) of DME were determined by Western blot analysis. The graphs show the levels of sAPPβ (B) and β-secretase (C), compared to the DMSO-treated control group. Values are expressed as a percentage of DMSO-treated control group. All data represent the means ± SD of three different experiments. * p < 0.05, significantly different from DMSO-treated control group (CTR: DMSO-treated control, DME: ethanol extract of D. mariesii roots, PC: positive control).

Figure 2

Effects of the solvent-partitioned fractions on the production of sAPPβ and the level of β-secretase. (A) The levels of sAPPβ and β-secretase from APP–CHO cells treated with 50 μg/mL of the solvent-partitioned fractions were determined by Western blot analysis. (B,C) The graphs show the levels of sAPPβ and β-secretase. Values are expressed as a percentage of DMSO-treated control group. All data represent the means ± SD of three different experiments. * p < 0.05, significantly different from DMSO-treated control group (Hx: n-hexane, DCM: dichloromethane, EA: ethyl acetate, DW: water).

Figure 3

Inhibitory effect of DME and solvent-partitioned fractions on Aβ aggregation/disaggregation. (A) Aβ was incubated with 100, 20, and 4 μg/mL of DME and solvent-partitioned fractions (Hx: n-hexane, DCM: dichloromethane, EA: ethyl acetate, DW: water). After 24 h, the Aβ aggregation was determined by Th T assay. (B) Aβ pre-aggregated for 24 h was incubated with DME and solvent-partitioned fractions (100, 20, and 4 μg/mL). After additional 24 h, the Aβ aggregation was determined by Th T assay. All data represent the means ± SD of three different experiments, * p < 0.05, significantly different from Aβ-only group.

Figure 4

The chemical structures of the isolated compounds from the ethyl acetate fraction of DME.

Figure 5

The effects of compounds 1–4 on the production of sAPPβ and the level of β-secretase. (A,D) The levels of sAPPβ and β-secretase from APP–CHO cells treated with compounds 1–4 (50 and 10 μg/mL) were determined by Western blot analysis. (B,C,E,F) The graphs show the levels of sAPPβ and β-secretase. Values are expressed as a percentage of DMSO-treated control group. All data represent the means ± SD of three different experiments. * p < 0.05, significantly different from DMSO-treated control group.

Figure 6

Inhibitory effect of isolated compounds on Aβ aggregation/disaggregation. (A) Aβ was incubated with 50 and 10 μg/mL of compounds isolated from DME. After 24 h, the Aβ aggregation was determined by Th T assay. (B) Aβ pre-aggregated for 24 h was incubated with 50 and 10 μg/mL of compounds isolated from DME. After 24 h, the Aβ disaggregation was determined by Th T assay. All data represent the means ± SD of three different experiments. * p < 0.05, significantly different from Aβ-only group.

Figure 7

Effect of DME on scopolamine-induced memory impairment in mice. (A) A schematic illustration of the animal study. (B) The body weight of the mice was measured twice a week for 4 weeks. (C) The retention time staying in the dark room of the mice in the passive avoidance test was measured. (D) The escape latency, i.e., the time for the mice to find the escape platform in the Morris water maze test, was recorded up to 120 s. All data represent the mean ± SE (n = 9). * p < 0.05, # p < 0.005, significantly different from scopolamine-treated control groups (N; normal, C; saline + scopolamine, P; donepezil + scopolamine, DME-L; 200 mg/kg + scopolamine, DME-H; 500 mg/kg + scopolamine).

Figure 8

Effect of DME on the levels of ACh and AChE in mice. (A) The levels of ACh in the whole brains was determined by ELISA. (B) The activity of AChE in the whole brains of mice was determined by ELISA. All data represent the mean ± SE (n = 9). * p < 0.05, # p < 0.005, significantly different from scopolamine-treated control groups (N; normal, C; saline + scopolamine, P; donepezil + scopolamine, DME-L; 200 mg/kg + scopolamine, DME-H; 500 mg/kg + scopolamine).

Figure 9

Effect of DME on Aβ deposition, BDNF, and CREB in the brains. Representative images of immunohistochemical analysis with anti-Aβ antibody (A), and anti-BDNF, anti-CREP, and anti-p-CREB antibodies (B) in the hippocampal region are shown (n = 7). Aβ depositions are indicated by arrows. (C) Immune-positive cells with BDNF, CREB, and p-CREB were quantified with Image J Software (1.5k version). Scale bar = 100 μm. All data represent the mean ± SE (n = 7). * p < 0.05, # p < 0.005, significantly different from scopolamine-treated control groups (N; normal, C; saline + scopolamine, P; donepezil + scopolamine, DME-L; 200 mg/kg + scopolamine, DME-H; 500 mg/kg + scopolamine).