Mixture of Phlorotannin and Fucoidan from Ecklonia cava Prevents the Aβ-Induced Cognitive Decline with Mitochondrial and Cholinergic Activation

Abstract

The anti-amnesic effect of a mixture (4:6 = phlorotannin:fucoidan from Ecklonia cava, P4F6) was evaluated on amyloid-beta peptide (Aβ)-induced cognitive deficit mice. The cognitive function was examined by Y-maze, passive avoidance, and Morris water maze tests, and the intake of the mixture (P4F6) showed an ameliorating effect on Aβ-induced learning and memory impairment. After the behavioral tests, superoxide dismutase (SOD) activity and thiobarbituric acid-reactive substances (TBARS) contents were confirmed in brain tissue, and in the results, the mixture (P4F6) attenuated Aβ-induced oxidative stress. In addition, mitochondrial activity was evaluated by mitochondrial reactive oxygen species (ROS) content, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content, and mitochondria-mediated apoptotic signaling pathway, and the mixture (P4F6) enhanced mitochondrial function. Furthermore, the mixture (P4F6) effectively regulated tau hyperphosphorylation by regulating the protein kinase B (Akt) pathway, and promoted brain-derived neurotrophic factor (BDNF) in brain tissue. Moreover, in the cholinergic system, the mixture (P4F6) ameliorated acetylcholine (ACh) content by regulating acetylcholinesterase (AChE) activity and choline acetyltransferase (ChAT) expression in brain tissue. Based on these results, we suggest that this mixture of phlorotannin and fucoidan (P4F6) might be a substance for improving cognitive function by effectively regulating cognition-related molecules.

Keywords: Ecklonia cava; amyloid-beta; cholinergic function; cognition; fucoidan; mitochondrial function; phlorotannin; tau phosphorylation.

Figure 1

Ameliorating effect of mixture (4:6 = phlorotannin:fucoidan from Ecklonia cava, P4F6) on Aβ-induced learning and memory impairment. The spontaneous alternation behavior and number of arm entries (a) in the Y-maze test. Step-through latency time in the passive avoidance test (b). Escape latency in the hidden platform (c), time in the W zone for the probe trial (d) in the Morris water maze test. Values are mean ± SD (n = 9), and different small letters^(a–d) above the columns in the figures indicate statistical difference between each group (ANOVA and Duncan’s new multiple-range tests; p < 0.05).

Figure 1

Ameliorating effect of mixture (4:6 = phlorotannin:fucoidan from Ecklonia cava, P4F6) on Aβ-induced learning and memory impairment. The spontaneous alternation behavior and number of arm entries (a) in the Y-maze test. Step-through latency time in the passive avoidance test (b). Escape latency in the hidden platform (c), time in the W zone for the probe trial (d) in the Morris water maze test. Values are mean ± SD (n = 9), and different small letters^(a–d) above the columns in the figures indicate statistical difference between each group (ANOVA and Duncan’s new multiple-range tests; p < 0.05).

Figure 2

Antioxidant effect of mixture (4:6 = phlorotannin:fucoidan from Ecklonia cava, P4F6) on Aβ-induced oxidative stress. SOD (a) and TBARS (b) contents in brain tissue. Values are mean ± SD (n = 9), and different small letters (^a–c) above the columns in the figures indicate statistical difference between each group (ANOVA and Duncan’s new multiple-range tests; p < 0.05).

Figure 3

Mitochondrial activity of the mixture (4:6 = phlorotannin:fucoidan from Ecklonia cava, P4F6) on Aβ-induced mitochondrial damage. Mitochondrial ROS content (a), mitochondrial membrane potential (MMP) (b), and ATP content (c) on mitochondria isolated from brain tissue. Band images of Western blot analysis (d), the expression level of mitochondria-dependent apoptotic molecules (BAX, cytosolic cytochrome c, and caspase-3) (e) in mouse brain tissue. Values are mean ± SD (n = 5), and different small letters^(a–d) above the columns in the figures indicate statistical difference between each group (ANOVA and Duncan’s new multiple-range tests; p < 0.05).

Figure 3

Mitochondrial activity of the mixture (4:6 = phlorotannin:fucoidan from Ecklonia cava, P4F6) on Aβ-induced mitochondrial damage. Mitochondrial ROS content (a), mitochondrial membrane potential (MMP) (b), and ATP content (c) on mitochondria isolated from brain tissue. Band images of Western blot analysis (d), the expression level of mitochondria-dependent apoptotic molecules (BAX, cytosolic cytochrome c, and caspase-3) (e) in mouse brain tissue. Values are mean ± SD (n = 5), and different small letters^(a–d) above the columns in the figures indicate statistical difference between each group (ANOVA and Duncan’s new multiple-range tests; p < 0.05).

Figure 4

The regulating effect of the mixture (4:6 = phlorotannin:fucoidan from Ecklonia cava, P4F6) on Aβ-induced cognitive dysfunction. Band image of Western blot analysis (a), tau phosphorylation-related molecules (p-JNK, p-AKT, p-GSK-3β, and p-Tau) (b) in mouse brain tissue. Values are mean ± SD (n = 5), and different small letters (^a,b) above the columns in the figures indicate statistical difference between each group (ANOVA and Duncan’s new multiple-range tests; p < 0.05).

Figure 5

Effect of of the mixture (4:6 = phlorotannin:fucoidan from Ecklonia cava, P4F6) on Aβ-induced cholinergic dysfunction. AChE activity (a), band images and expression level of ChAT and AChE (b), and ACh contents (c) in mouse brain tissue. Values are mean ± SD (n = 9), and different small letters (^a–d) above the columns in the figures indicate statistical difference between each group (ANOVA and Duncan’s new multiple-range tests; p < 0.05).

Figure 5

Effect of of the mixture (4:6 = phlorotannin:fucoidan from Ecklonia cava, P4F6) on Aβ-induced cholinergic dysfunction. AChE activity (a), band images and expression level of ChAT and AChE (b), and ACh contents (c) in mouse brain tissue. Values are mean ± SD (n = 9), and different small letters (^a–d) above the columns in the figures indicate statistical difference between each group (ANOVA and Duncan’s new multiple-range tests; p < 0.05).