Ameliorative Effects of Rhoifolin in Scopolamine-Induced Amnesic Zebrafish (Danio rerio) Model

Abstract

Rhoifolin (Rho) exerts many biological activities such as anticancer, antidiabetic, hepatoprotective, antirheumatic, antibacterial, and antiviral properties. The neuroprotective action of this compound has not been studied. The goal of this study was to investigate the improvement impact of Rho on scopolamine (Sco)-induced zebrafish anxiety, amnesia, and brain oxidative stress and to elucidate the underlying mechanisms involved. Zebrafish were treated with Rho (1, 3, and 5 μg/L) for nine consecutive days and were subsequently subjected to Sco (100 μM) 30 min before behavioral tests (novel tank diving test, Y-maze, and novel object recognition tests). Rho was isolated from Chorisia crispiflora (Malvaceae) leaves and identified by different spectroscopic techniques. To further assess the possible mechanisms of Rho in enhancing the memory capacities in zebrafish, the in vivo antioxidant status and acetylcholinesterase (AChE) activity was also evaluated. Rho from Chorisia crispiflora leaves was identified. Rho could alleviate anxiety, memory deficits, and brain oxidative stress in Sco-treated zebrafish and could regulate the cholinergic function by inhibiting the AChE activity. Our results demonstrated that Rho could be a promising candidate compound against anxiety and amnesia by restoring the cholinergic activity and the amelioration of brain oxidative stress.

Keywords: anxiety; cholinergic function; memory; oxidative stress; rhoifolin; zebrafish.

Figure 1

Chemical structure of rhoifolin (apigenin 7-O-β neohesperidoside).

Figure 2

Rhoifolin (Rho: 1, 3, and 5 μg/L) improved the locomotion pattern and reduced anxiety in the novel tank diving test (NTT). (A) Representative locomotion tracking pattern of the control, scopolamine (Sco: 100 μM), rhoifolin (Rho: 1, 3, and 5 µg/L), and imipramine (IMP: 20 mg/L) treated groups. (B) Representation of the time spent in the top zone by zebrafish in the tank in different groups. (C) Representation of the time spent in top/bottom ratio in different groups. (D) Representation of the total distance traveled by zebrafish in the tank in different groups. (E) Representation of the distance top/bottom ratio in different groups. The values are means ± S.E.M. (n = 10). For Tukey’s post hoc analyses: (B) Control vs. Sco (100 μM): ### p < 0.0001, Sco (100 μM) vs. Rho (1 μg/L): ## p < 0.001, Sco (100 μM) vs. Rho (3 μg/L): ### p < 0.0001 and Sco (100 μM) vs. Rho (5 μg/L): ### p < 0.0001; (C) Control vs. Sco (100 μM): ### p < 0.0001, Sco (100 μM) vs. Rho (1 μg/L): # p < 0.01, Sco (100 μM) vs. Rho (3 μg/L): ## p < 0.001, and Sco (100 μM) vs. Rho (5 μg/L): ### p < 0.0001; (D) Control vs. Sco (100 μM): ### p < 0.0001, Sco (100 μM) vs. Rho (1 μg/L): ### p < 0.0001, Sco (100 μM) vs. Rho (3 μg/L): ### p < 0.0001, and Sco (100 μM) vs. Rho (5 μg/L): ### p < 0.0001; (E) Control vs. Sco (100 μM): ### p < 0.0001, Sco (100 μM) vs. Rho (1 μg/L): # p < 0.01, Sco (100 μM) vs. Rho (3 μg/L): ### p < 0.0001, and Sco (100 μM) vs. Rho (5 μg/L): ### p < 0.0001.

Figure 3

Rhoifolin (Rho: 1, 3, and 5 μg/L) improved the locomotion pattern and memory in the Y-maze test. (A) Representative locomotion tracking pattern of the control, scopolamine (Sco: 100 µM), rhoifolin (Rho: 1, 3, and 5 µg/L) and galantamine (GAL: 1 mg/L) treated groups. (B) Representation of the time spent in each arm (start, other, and novel arm) in different groups. (C) Representation of the total distance traveled by zebrafish in the tank in different groups. (D) Representation of the turn angle of zebrafish in the tank in different groups. Values are means ± S.E.M. (n = 10). For Tukey’s post hoc analyses: (B) Control vs. Sco (100 µM): ### p < 0.0001, Sco vs. Rho (1 µg/L): ### p < 0.0001, Sco vs. Rho (3 µg/L): ### p < 0.0001, and Sco vs. Rho (5 µg/L): ### p < 0.0001; (C) Control vs. Sco (100 µM): # p < 0.01, Sco vs. Rho (1 µg/L): ## p < 0.001, Sco vs. Rho (3 µg/L): ### p < 0.0001, and Sco vs. Rho (5 µg/L): ### p < 0.0001; (D) Control vs. Sco (100 µM): ### p < 0.0001, Sco vs. Rho (1 µg/L): ### p < 0.0001, Sco vs. Rho (3 µg/L): ## p < 0.0001, and Sco vs. Rho (5 µg/L): ## p < 0.0001.

Figure 4

Rhoifolin (Rho: 1, 3, and 5 μg/L) improved memory in the novel object recognition test (NOR). (A) Representative locomotion tracking pattern of the control, scopolamine (Sco: 100 µM), rhoifolin (Rho: 1, 3, and 5 µg/L), and galantamine (GAL: 1 mg/L) treated groups. (B) Representation of the percentages of preference in different groups. (C) Representation of the exploratory time in different groups. Values are means ± S.E.M. (n = 10). For Tukey’s post hoc analyses: (B) Control vs. Sco (100 µM): # p < 0.01, Sco vs. Rho (3 µg/L): # p < 0.01, and Sco vs. Rho (5 µg/L): # p < 0.01; (C) Control vs. Sco (100 µM): # p < 0.01, Sco vs. Rho (1 µg/L): ### p < 0.0001, Sco vs. Rho (3 µg/L): ### p < 0.0001, and Sco vs. Rho (5 µg/L): ### p < 0.0001.

Figure 5

Rhoifolin (Rho: 1, 3, and 5 μg/L) exhibited an anti-AChE effect and improved the antioxidant status in the zebrafish brain. (A–D) Representation of the enzymes’ specific activity (AChE, SOD, CAT, and GPX) in different groups; (E,F) Representation of the protein carbonyl and MDA levels in different groups. Values are means ± S.E.M. (n = 10). For Tukey’s post hoc analyses: (A) Control vs. Sco (100 µM): ### p < 0.0001, Sco vs. Rho (1 µg/L): ## p < 0.001, Sco vs. Rho (3 µg/L): ## p < 0.001 and Sco vs. Rho (5 µg/L): ## p < 0.001; (B) Control vs. Sco (100 µM): ### p < 0.0001, Sco vs. Rho (1 µg/L): # p < 0.01, Sco vs. Rho (3 µg/L): ## p < 0.001 and Sco vs. Rho (5 µg/L): ### p < 0.0001; C. Control vs. Sco (100 µM): ### p < 0.0001, Sco vs. Rho (1 µg/L): ## p < 0.001, Sco vs. Rho (3 µg/L): ### p < 0.0001 and Sco vs. Rho (5 µg/L): ### p < 0.0001; (D) Control vs. Sco (100 µM): ### p < 0.0001, Sco vs. Rho (1 µg/L): ## p < 0.001, Sco vs. Rho (3 µg/L): ### p < 0.0001 and Sco vs. Rho (5 µg/L): ### p < 0.0001; (E) Control vs. Sco (100 µM): ## p < 0.001, Sco vs. Rho (3 µg/L): ## p < 0.001 and Sco vs. Rho (5 µg/L): ## p < 0.001; and (F) Control vs. Sco (100 µM): ## p < 0.001, Sco vs. Rho (1 µg/L): ## p < 0.001, Sco vs. Rho (3 µg/L): ## p < 0.001 and Sco vs. Rho (5 µg/L): ### p < 0.0001.