The effects of cyanidin derivatives on locomotor impairments of AD zebrafish. (A) The chemical structure and abbreviations of cyanidin derivatives. (B) A representative image of the larvae movement track from each group. In the heat map, the colors red, green, and blue are used to represent fast, medium-speed, and slow actions, respectively. (C) Distance travelled in mm. (D) Average speed in mm/s. ## p < 0.01 vs. Con group, * p < 0.05 and ** p < 0.01 vs. model group. Group notes: Control: control group, Mod: model group (AlCl₃), Dpz: donepezil + AlCl₃ group, C3GG: cyanidin-3-O-(trans-p-coumaroyl)-diglycoside + AlCl₃ group, C3R: cyanidin-3-O-rutinoside + AlCl₃ group, C3A: cyanidin-3-O-arabinoside + AlCl₃ group, C3S: cyanidin-3-O-sophoroside, + AlCl₃ group, C3X: cyanidin-3-O-xyloside+ AlCl₃ group.

The effects of cyanidin derivatives on the dysfunction of cholinergic system and oxidative stress of AD zebrafish. (A) AChE activity. (B) SOD activity. (C) GSH level. (D) MDA level. ## p < 0.01 vs. Con group, * p < 0.05 and ** p < 0.01 vs. model group. Group notes: Control: control group, Mod: model group (AlCl₃), Dpz: donepezil + AlCl₃ group, C3GG: cyanidin-3-O-(trans-p-coumaroyl)-diglycoside + AlCl₃ group, C3R: cyanidin-3-O-rutinoside + AlCl₃ group, C3A: cyanidin-3-O-arabinoside + AlCl₃ group, C3S: cyanidin-3-O-sophoroside, + AlCl₃ group, C3X: cyanidin-3-O-xyloside+ AlCl₃ group.

The effects of cyanidin derivatives on the transcriptional alterations of AD-related genes of AD zebrafish. (A) Relative mRNA expression of ache. (B) Relative mRNA expression of tau. (C) Relative mRNA expression of appb. (D) Relative mRNA expression of aβ. ## p < 0.01 vs. Con group, ** p < 0.01 vs. model group. Group notes: Control: control group, Mod: model group (AlCl₃), Dpz: donepezil + AlCl₃ group, C3GG: cyanidin-3-O-(trans-p-coumaroyl)-diglycoside + AlCl₃ group, C3R: cyanidin-3-O-rutinoside + AlCl₃ group, C3A: cyanidin-3-O-arabinoside + AlCl₃ group, C3S: cyanidin-3-O-sophoroside, + AlCl₃ group, C3X: cyanidin-3-O-xyloside+ AlCl₃ group.

Comparison of histopathological alterations in brain tissues among various treatment groups. (A) Normal histological section of larval brain (40×). (B) Larvae brain exposed to 80 μM AlCl₃ (40×). (C) Larvae brain exposed to AlCl₃ + 4μM Dpz (40×). (D) Larvae brain exposed to AlCl₃ + 4μM C3GG (40×). (E) Larvae brain exposed to AlCl₃ + 4μM C3R (40×). (F) Larvae brain exposed to AlCl₃ + 4μM C3A (40×). (G) Larvae brain exposed to AlCl₃ + 4μM C3S (40×). (H) Larvae brain exposed to AlCl₃ + 4μM C3X (40×). (I) Neuronal density in zebrafish brain. (J) semi-quantitative analysis scores in H&E staining. ## p < 0.01 vs. Con group, * p < 0.05 and ** p < 0.01 vs. model group. (The neurons are highlighted by yellow arrows. Red arrows indicate a small population of neurons exhibiting pyknosis and deeply stained necrosis, whereas green arrows point to areas of midbrain dilation accompanied by balloon-like degeneration).

Comparison of Nissl staining of neural cells in brain tissues among various treatment groups. Group notes: Control: control group, Mod: model group (AlCl₃), Dpz: donepezil + AlCl₃ group, C3GG: cyanidin-3-O-(trans-p-coumaroyl)-diglycoside + AlCl₃ group, C3R: cyanidin-3-O-rutinoside + AlCl₃ group, C3A: cyanidin-3-O-arabinoside + AlCl₃ group, C3S: cyanidin-3-O-sophoroside, + AlCl₃ group, C3X: cyanidin-3-O-xyloside+ AlCl₃ group. (A) Nissl staining diagram of the normal group (40×). (B) Nissl staining diagram of the model group (40×). (C) Nissl staining diagram of the Dpz group (40×). (D) Nissl staining diagram of the C3GG group (40×). (E) Nissl staining diagram of the C3R group (40×). (F) Nissl staining diagram of the C3A group (40×). (G) Nissl staining diagram of the C3S group (40×). (H) Nissl staining diagram of the C3X group (40×). (I) The number of Nissl bodies in the zebrafish brain. ## p < 0.01 vs. Con group, ** p < 0.01 vs. model group.

Molecular docking results of AChE and cyanidin derivatives. (A) C3GG. (B) C3R. (C) C3A. (D) C3S. (E) C3X. (F) Binding affinity between AChE and cyanidin derivatives.

Visualization of results from molecular dynamics simulations. (A) RMSD. (B) RMSF. (C) Rg. (D) H-Bonds Number. (E) SASA.

Molecular dynamics simulation results. (A) The free energy landscape map of cyanidin derivatives. (B) Structural changes of the AChE-cyanidin derivatives complex at different time points. The colors red, green, blue, yellow, and orange, respectively, represent the structural comparisons of molecular dynamics simulations at 0, 25, 50, 75, and 100 ns. (C) Free energy of cyanidin derivatives binding to AChE calculated by MM/GBSA method. VDWAALS, EEL, EGB, ESURF, GGAS, GSOLV, and TOTAL represent van der Waals forces, electrostatic potential energy, polar solvation energy, nonpolar solvation energy, molecular mechanics term, solvation energy term, and binding free energy, respectively.