Cytotoxicity of different procyanidins in PC12 cells. (a) Survival of PC12 cells in the C treatment group; (b) Survival rate of PC12 cells in the EC treatment group; (c) Survival rate of PC12 cells in the ECG treatment group; (d) Survival rate of PC12 cells in the B1 treatment group; (e) Survival rate of PC12 cells in the B2 treatment group; (f) Survival rate of PC12 cells in the B3 treatment group; (g) Survival rate of PC12 cells in the B4 treatment group; (h) Survival rate of PC12 cells in the B1-G treatment group; (i) Survival rate of PC12 cells in the B2-G treatment group; (j) Survival rate of PC12 cells in the C1 treatment group. Data are expressed as the mean ± SD. All experiments were conducted six times. Values with different letters above each bar represent significant differences (p < 0.05, one-way ANOVA). PC12, rat pheochromocytoma cell line; EC, epicatechin; ECG, epicatechin gallate; B1, procyanidin B1; B2, procyanidin B2; B3, procyanidin B3; B4, procyanidin B4; B1-G, procyanidin B1-3-O-gallate; B2-G: procyanidin B2-3-O-gallate; C1: procyanidin C1; SD, standard deviation; ANOVA, one-way analysis of variance.

Cytotoxicity of different procyanidins in PC12 cells. (a) Survival of PC12 cells in the C treatment group; (b) Survival rate of PC12 cells in the EC treatment group; (c) Survival rate of PC12 cells in the ECG treatment group; (d) Survival rate of PC12 cells in the B1 treatment group; (e) Survival rate of PC12 cells in the B2 treatment group; (f) Survival rate of PC12 cells in the B3 treatment group; (g) Survival rate of PC12 cells in the B4 treatment group; (h) Survival rate of PC12 cells in the B1-G treatment group; (i) Survival rate of PC12 cells in the B2-G treatment group; (j) Survival rate of PC12 cells in the C1 treatment group. Data are expressed as the mean ± SD. All experiments were conducted six times. Values with different letters above each bar represent significant differences (p < 0.05, one-way ANOVA). PC12, rat pheochromocytoma cell line; EC, epicatechin; ECG, epicatechin gallate; B1, procyanidin B1; B2, procyanidin B2; B3, procyanidin B3; B4, procyanidin B4; B1-G, procyanidin B1-3-O-gallate; B2-G: procyanidin B2-3-O-gallate; C1: procyanidin C1; SD, standard deviation; ANOVA, one-way analysis of variance.

Effect of different procyanidins on the survival of PC12 cells induced by H2O2. (a) Effect of 2.5 μM procyanidins on the survival of PC12 cells induced by H2O2; (b) Effect of 5 μM procyanidins on the survival of PC12 cells induced by H2O2. Control, Blank control group; Model. H2O2 (200 μM); Positive control, NAC (20 μM) + H2O2 (200 μM); C, C + H2O2 (200 μM); EC, EC + H2O2 (200 μM); ECG, ECG + H2O2 (200 μM); B1, B1 + H2O2 (200 μM); B2, B2 + H2O2 (200 μM); B3, B3 + H2O2 (200 μM); B4, B4 + H2O2 (200 μM); B1-G, B1-G + H2O2 (200 μM); B2-G, B2-G + H2O2 (200 μM); C1, C1 + H2O2 (200 μM). Data are expressed as the mean ± SD. All experiments were conducted six times. Values with different letters above each bar represent significant differences (p < 0.05, one-way ANOVA). NAC, N-acetyl-l-cysteine.

Effects of procyanidins with different structures on oxidative stress in PC12 cells treated with H₂O₂. (a) Representative fluorescence photomicrographs of PC12 cells; (b) ROS levels; (c) MDA levels; (d) GSH-Px activity; (e) CAT activity; (f) SOD activity. Data are expressed as the mean ± SD. Control, Blank control group; Model, H₂O₂ (200 μM); Positive control, NAC (20 μM) + H₂O₂ (200 μM); C, C (5 μM) + H₂O₂ (200 μM); EC, EC (5 μM) + H₂O₂ (200 μM); ECG, ECG (5 μM) + H₂O₂ (200 μM); B1, B1 (5 μM) + H₂O₂ (200 μM); B2, B2 (5 μM) + H₂O₂ (200 μM); B3, B3 (5 μM) + H₂O₂ (200 μM); B4, B4 (5 μM) + H₂O₂ (200 μM); B1-G, B1-G (5 μM) + H₂O₂ (200 μM); B2-G, B2-G (5 μM) + H₂O₂ (200 μM); C1, C1 (5 μM) + H₂O₂ (200 μM). All experiments were conducted three times. Values with different letters are significantly different (p < 0.05, one-way ANOVA). ROS, reactive oxygen species; MDA, malondialdehyde; GSH-Px, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase.

Effects of procyanidins with different structures on oxidative stress in PC12 cells treated with H₂O₂. (a) Representative fluorescence photomicrographs of PC12 cells; (b) ROS levels; (c) MDA levels; (d) GSH-Px activity; (e) CAT activity; (f) SOD activity. Data are expressed as the mean ± SD. Control, Blank control group; Model, H₂O₂ (200 μM); Positive control, NAC (20 μM) + H₂O₂ (200 μM); C, C (5 μM) + H₂O₂ (200 μM); EC, EC (5 μM) + H₂O₂ (200 μM); ECG, ECG (5 μM) + H₂O₂ (200 μM); B1, B1 (5 μM) + H₂O₂ (200 μM); B2, B2 (5 μM) + H₂O₂ (200 μM); B3, B3 (5 μM) + H₂O₂ (200 μM); B4, B4 (5 μM) + H₂O₂ (200 μM); B1-G, B1-G (5 μM) + H₂O₂ (200 μM); B2-G, B2-G (5 μM) + H₂O₂ (200 μM); C1, C1 (5 μM) + H₂O₂ (200 μM). All experiments were conducted three times. Values with different letters are significantly different (p < 0.05, one-way ANOVA). ROS, reactive oxygen species; MDA, malondialdehyde; GSH-Px, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase.

Effects of procyanidins with different structures on the Nrf2/ARE pathway in PC12 cells treated with H2O2. (a) Protein levels of Nrf2, as determined by Western blotting; (b) Relative Nrf2/GAPDH protein expression (ratio to control); (c) Protein expression levels of nuclear Nrf2 and cytoplasmic Nrf2, as determined by Western blotting; (d) Relative nuclear Nrf2/Lamin B protein expression (ratio to control); (e) Relative cytoplasmic Nrf2/GAPDH protein expression (ratio to control); (f) Protein levels of HO-1 and NQO1, as determined by Western blotting; (g) Relative HO-1/GAPDH protein expression (ratio to control); (h) Relative NQO1/GAPDH protein expression (ratio to control). Data are expressed as the mean ± SD. Control, Blank control group; Model, H2O2 (200 μM); Positive control, NAC (20 μM) + H2O2 (200 μM); C, C (5 μM) + H2O2 (200 μM); EC, EC (5 μM) + H2O2 (200 μM); ECG, ECG (5 μM) + H2O2 (200 μM); B1, B1 (5 μM) + H2O2 (200 μM); B2, B2 (5 μM) + H2O2 (200 μM); B3, B3 (5 μM) + H2O2 (200 μM); B4, B4 (5 μM) + H2O2 (200 μM); B1-G, B1-G (5 μM) + H2O2 (200 μM); B2-G, B2-G (5 μM) + H2O2 (200 μM); C1, C1 (5 μM) + H2O2 (200 μM). All experiments were conducted three times. Values with different letters are significantly different (p < 0.05, one-way ANOVA). Nrf2, nuclear factor-erythroid 2-related factor 2; ARE, antioxidant response element; HO-1, heme oxygenase 1; NQO1, NAD(P)H: quinone oxidoreductase 1.

Verification of the role of Nrf2 in the protective effect of procyanidins with different structures. (a) Knockout efficiency was confirmed by determination of Nrf2 protein expression using Western blotting; (b) Relative Nrf2/GAPDH protein expression (ratio to control); (c) Cell viability. Data are shown as the mean ± SD. All experiments were conducted three times. Values with different letters are significantly different (p < 0.05, one-way ANOVA).

Effects of procyanidins with different structures on exercise ability in zebrafish treated with H2O2. (a) Swimming traces of zebrafish in each group; (b) Average total distance of zebrafish in each group; (c) Swimming traces of zebrafish in each group; (d) Average total distance of zebrafish in each group. Data are expressed as the mean ± SD. Control, Blank control group; Model, H2O2 (300 μM); Positive control, NAC (30 μM) + H2O2 (300 μM); C, C (25 μM) + H2O2 (300 μM); EC, EC (25 μM) + H2O2 (300 μM); ECG, ECG (25 μM) + H2O2 (300 μM); B1, B1 (25 μM) + H2O2 (300 μM); B2, B2 (25 μM) + H2O2 (300 μM); B3, B3 (25 μM) + H2O2 (300 μM); B4, B4 (25 μM) + H2O2 (300 μM); B1-G, B1-G (25 μM) + H2O2 (300 μM); B2-G, B2-G (25 μM) + H2O2 (300 μM); C1, C1 (25 μM) + H2O2 (300 μM). All experiments were conducted three times. Values with different letters are significantly different (p < 0.05, one-way ANOVA).

Effects of procyanidins with different structures on oxidative stress in zebrafish treated with H2O2. (a) Representative fluorescence photomicrographs of zebrafish; (b) ROS levels; (c) MDA levels; (d) GSH-Px activity; (e) CAT activity; (f) SOD activity. Data are expressed as the mean ± SD. Control, Blank control group; Model, H2O2 (300 μM); Positive control, NAC (30 μM) + H2O2 (300 μM); C, C (25 μM) + H2O2 (300 μM); EC, EC (25 μM) + H2O2 (300 μM); ECG, ECG (25 μM) + H2O2 (300 μM); B1, B1 (25 μM) + H2O2 (300 μM); B2, B2 (25 μM) + H2O2 (300 μM); B3, B3 (25 μM) + H2O2 (300 μM); B4, B4 (25 μM) + H2O2 (300 μM); B1-G, B1-G (25 μM) + H2O2 (300 μM); B2-G, B2-G (25 μM) + H2O2 (300 μM); C1, C1 (25 μM) + H2O2 (300 μM). All experiments were conducted three times. Values with different letters are significantly different (p < 0.05, one-way ANOVA).

Effects of procyanidins with different structures on oxidative stress in zebrafish treated with H2O2. (a) Representative fluorescence photomicrographs of zebrafish; (b) ROS levels; (c) MDA levels; (d) GSH-Px activity; (e) CAT activity; (f) SOD activity. Data are expressed as the mean ± SD. Control, Blank control group; Model, H2O2 (300 μM); Positive control, NAC (30 μM) + H2O2 (300 μM); C, C (25 μM) + H2O2 (300 μM); EC, EC (25 μM) + H2O2 (300 μM); ECG, ECG (25 μM) + H2O2 (300 μM); B1, B1 (25 μM) + H2O2 (300 μM); B2, B2 (25 μM) + H2O2 (300 μM); B3, B3 (25 μM) + H2O2 (300 μM); B4, B4 (25 μM) + H2O2 (300 μM); B1-G, B1-G (25 μM) + H2O2 (300 μM); B2-G, B2-G (25 μM) + H2O2 (300 μM); C1, C1 (25 μM) + H2O2 (300 μM). All experiments were conducted three times. Values with different letters are significantly different (p < 0.05, one-way ANOVA).

Effects of procyanidins with different structures on the Nrf2/ARE pathway in zebrafish treated with H2O2. (a) Nrf2 levels; (b) NQO1 levels; (c) HO-1 levels. Data are expressed as the mean ± SD. Control, Blank control group; Model, H2O2 (300 μM); Positive control, NAC (30 μM) + H2O2 (300 μM); C, C (25 μM) + H2O2 (300 μM); EC, EC (25 μM) + H2O2 (300 μM); ECG, ECG (25 μM) + H2O2 (300 μM); B1, B1 (25 μM) + H2O2 (300 μM); B2, B2 (25 μM) + H2O2 (300 μM); B3, B3 (25 μM) + H2O2 (300 μM); B4, B4 (25 μM) + H2O2 (300 μM); B1-G, B1-G (25 μM) + H2O2 (300 μM); B2-G, B2-G (25 μM) + H2O2 (300 μM); C1, C1 (25 μM) + H2O2 (300 μM). All experiments were conducted three times. Values with different letters are significantly different (p < 0.05, one-way ANOVA).