Oxidative stress promotes the production of reactive oxygen species (ROS), which include hydrogen peroxide (H₂O₂), superoxides (O₂•-), and hydroxyl radicals (HO•) [1]. Abnormally high levels of ROS in neurons can lead to mitochondrial dysfunction and oxidative damage, and oxidative damage leads to neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s disease, by increasing the permeability of the blood–brain barrier, tubulin alterations, and perturbation in synaptic transmissions [1–3]. In particular, H₂O₂ is thought to be a major precursor for ROS, and the accumulation of intracellular ROS in living cells accelerates mitochondrial dysfunction via the oxidation of DNA, proteins (e.g., Bcl-2 family and caspase family), and lipids [4].

Mitochondria have a potentially critical role in ageing and neurodegenerative diseases as important regulators of cell survival and neuron death [2]. Mitochondria are intracellular organelles that are central to energy metabolism in cells, but they account for more than 80% of the generation of ROS by respiratory chain complexes I and III. Therefore, mitochondrial dysfunction can cause serious diseases and health hazards [2, 5].

Some antioxidant enzymes and substances that have natural defense systems in living organisms have played an important role in inhibiting the cascade reaction of ROS production [6]. However, these are completely unable to remove oxidative stress or prevent oxidative damage. Antioxidants are useful materials that can delay and prevent cellular oxidative damage by scavenging ROS, activating defense systems against oxidative stress, and inhibiting the generation of ROS [6, 7]. Synthetic antioxidants including butylated hydroxytoluene, tert-butylhydroquinone, and propyl gallate are added for the purpose of inhibiting lipid peroxidation and improving stability [7]. However, their uses in food products are strictly limited owing to the potential health hazards of such compounds. There is an increasing need to replace these synthetic antioxidants with safe and effective natural antioxidants derived from natural foods. In addition, these natural antioxidants are known to protect the human body from oxidative damage and prevent the many chronic diseases that are associated with oxidative damage [8]. However, some natural antioxidants, such as phenolics and vitamin E, are difficult to use because they are water-insoluble. In addition, vitamin C, which is water-soluble, has the disadvantage that it is highly heat-sensitive and easily denatured [9]. Therefore, there is a need for an antioxidant that is both water-soluble and heat-stable.

In this regard, seaweed has been raising considerable interest and evaluated as an important material as a source of valuable functional metabolites [10]. Seaweeds have been widely used in various fields, such as food sources, feed, medicine, and energy-rich sources, and have been evaluated as extraordinary sustainable resources [10, 11]. Ecklonia cava, which is found mainly in Korea and Japan, is a species of brown algae that is an edible seaweed [11]. Two representative chemical compounds are generally found in brown algae: polysaccharides and polyphenols [9]. The polyphenols found in E. cava include phlorotannins (dieckol, 6,6-bieckol, eckol, and eckstolonol) and have reported antioxidant, anti-inflammatory, metalloproteinase-inhibitory, and bactericidal activities [11, 12]. Fucoidan was also found mainly in brown seaweed as a type of polysaccharide, and a substantial percentage was composed of L-fucose and sulfate ester groups [13]. Their structures indicated unique properties as secondary metabolites. In general, the reported biological activities of fucoidan have included anticoagulative, antitumor, and anti-inflammatory effects [11]. Therefore, there is great interest in fucoidan, as the biologically active component has highly considerable potential application in various fields, such as drugs, foods, and cosmetics, as functional materials. Unfortunately, the neuronal cell-protective effect of fucoidan, particularly of fucoidan derived from E. cava, has not yet been reported against H₂O₂-induced neuronal cell damage. Therefore, we have investigated the neuronal cell-protective effect of fucoidan extract by testing its regulation of mitochondrial function on hydrogen peroxide-induced oxidative damage in PC-12 cells.

Chemicals and Reagents

2,2’-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium persulfate, acetylthiocholine, 5,5’-dithiobis(2-nitro)benzoic acid (DTNB), trichloroacetic acid (TCA), thiobarbituric acid (TBA), dimethyl sulfoxide (DMSO), and all other chemicals used were purchased from Sigma-Aldrich Chemical Co. (USA). All chemicals used were of analytical grade.

Sample Preparation

Fucoidan extract from E. cava (FEC) was supplied by Seojin Biotech Co. Ltd. (Korea). Briefly, E. cava was hydrolyzed in 0.1 N HCl for 12 h at 45°C. This hydrolysate was filtered using a decanter (Hanil Sci-Med Co., Ltd., Korea) and immersed into ethanol (1:1) for 12 h. The immersed extract was centrifuged at 11,000 ×g, and the obtained pellet was freeze-dried. The freeze-dried pellet was used as the fucoidan extract and stored at -20°C for the experiments.

Antioxidant Activity Assessment

The free radical scavenging activity was investigated using an ABTS radical cation decolorization method [14]. ABTS radicals were produced by reacting 7 mM ABTS and potassium persulfate (2.45 mM) in 100 mM potassium phosphate buffer containing 150 mM NaCl (pH 7.4) at room temperature for more than 24 h prior to use. After more than 24 h, a stable ABTS radical solution was diluted with distilled water (A_{734 nm} = 0.700 ± 0.02). After this, the diluted ABTS radical solution (980 μl) was mixed with the sample (20 μl) and incubated for 10 min. After incubation, the absorbance was measured at 734 nm using a photospectrometer (UV-1201; Shimadzu, Japan), and the calculation was as follows:

ABTS radical scavenging (%) = [(control absorbance – sample absorbance)/control absorbance] × 100

The inhibitory effect of lipid peroxidation was evaluated using the MDA assay, which has been investigated as a modified method of Chang et al. [15]. The animal experimental process was approved by the Institutional Animal Care and Use Committee (IACUC) of Gyeongsang National University (certificate: GNU-170605-M0023). The brain tissue of ICR mice (4 weeks old, male) were homogenized in 20 mM Tris-HCl buffer (pH 7.4). The brain homogenate was centrifuged at 12,000 ×g for 10 min at 4°C. The supernatant was added to 10 μM FeSO₄ and 0.1 mM ascorbic acid and was incubated at 37°C for 1 h. After incubation, 30% TCA and 1% TBA were added to the mixture, which was then heated at 80°C for 20 min. After heating, the absorbance of the MDA-TBA complex was measured at 532 nm using a spectrophotometer (UV-1201; Shimadzu, Japan), and the calculation was as follows:

MDA inhibitory effect (%) = [(control absorbance – sample absorbance)/control absorbance] × 100

Cell Culture

Rat pheochromocytoma (PC-12) cells were purchased from the Korean Cell Line Bank (Republic of Korea), and incubated in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C and 5% CO₂ in a humidified atmosphere. MC-IXC, derived from human brain tissue cells, was purchased from American Type Culture Collection (USA), and was incubated in MEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C and 5% CO₂ in a humidified atmosphere.

Neuronal Cell-Protective Effect

The intracellular ROS content was measured using a 2’,7’-dichlorofluorescein diacetate (DCF-DA) assay. The PC-12 and MC-IXC cells were seeded in 96-well plates (0.5 × 10⁴/well), and incubated overnight. The samples were treated for 24 h, after which the sample and H₂O₂ groups but not the control group were treated with H₂O₂ (final concentration: 200 μM) for 3 h. DCF-DA (10 μM) was added to the pretreated cells for 50 min, and the produced DCF contents were quantified using a fluorometer (Infinite F200; Tecan, USA) at 485 nm (excitation wavelength) and 535 nm (emission wavelength) [16].

Cell viability against oxidative stress was measured using the MTT assay. After sampling and H₂O₂ treatment, an MTT solution in PBS (5 mg/ml) was added to each well and incubated for 2 h. Thereafter, the amount of violet MTT formazan crystals was measured using a microplate reader (EPOCH₂; BioTek, USA) at 570 nm (test wavelength) and 690 nm (reference wavelength) [16].

Measurement of Mitochondrial Activity

The mitochondrial membrane potential (MMP, ΔΨm) was measured using JC-1 dye as a lipophilic cationic probe. JC-1 shows fluorescence emission with a green fluorescent monomer (530 nm) at a depolarized membrane potential, and J-aggregates are formed via dislocation-dependent accumulation at the hyperpolarization potential, creating a red fluorescence emission (590 nm). JC-1 is represented by a fluorescence emission shift from 530 nm to 590 nm. After sample and H₂O₂ treatment, JC-1 solution (1 μM) was added to each well and they were incubated for 30 min. Thereafter, the fluorescence emission contents were measured using a fluorometer (Infinite F200; Tecan) at 485 nm (excitation wavelength) and each 530 and 590 nm (emission wavelength), and was calculated by the 590 nm/530 nm fluorescence emission ratio [17].

ATP levels were measured using a commercial kit (Promega, USA), based on the principle that luciferase produces light (ATP-dependent oxidation), the amount of which could be detected by a microplate luminometer (Promega).

Western Blot Analysis

Pretreated PC-12 cells were washed with cold PBS and lysed with RIPA buffer containing proteinase inhibitors on ice for 15 min. After 15 min, the lysed cells were centrifuged at 13,000 ×g for 10 min, and the protein contents were quantized by Bradford assay. The lysed sample including the equal protein was added to Laemmli buffer and denatured at 95°C for 5 min. The denatured protein was subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride membranes. The transferred membrane was blocked with 5% skim milk in TBST for 1 h and the then immunoblotted overnight at 4°C with primary antibodies, including phosphorylated AMP-activated protein kinase (p-AMPK), Bcl-2-associated X protein (BAX), and β-actin. The membrane immunoblotted by the primary antibody was incubated with the corresponding secondary antibodies for 90 min at room temperature. The membrane bands were visualized with One-Step Ultra TMB blotting solution (Thermo Scientific, USA), and the results were analyzed using ImageJ software (US National Institutes of Health, USA).

Acetylcholinesterase-Inhibitory Effect and Inhibitory Pattern Analysis

The inhibitory effect against acetylcholinesterase (AChE) was evaluated using Ellman’s photometric method [18]. AChE derived from cholinergic neurons was extracted by homogenizing PC-12 cells with extraction buffer (10 mM Tris-HCl (pH 7.2) buffer containing 1% Triton X-100, 50 mM MgCl₂, and 1 M NaCl) using a Bullet Blender Storm (Next Advance, USA). The homogenized cells were centrifuged at 10,000 ×g for 30 min, and the supernatant was used as an AChE extract. The samples in 50 mM sodium phosphate buffer were mixed with enzymes and incubated for 15 min. The incubated mixtures were added to Ellman’s reaction mixture (0.5 mM acetylthiocholine and 1 mM DTNB in 50 mM sodium phosphate buffer (pH 8.0)) and incubated for 10 min, after which the mixtures were read at 405 nm using the microplate reader (EPOCH₂; BioTek). Inhibitory pattern analysis against AChE was determined by incubating the mixtures of AChE and samples for 15 min and then treating each with Ellman’s reaction mixture as substrate at 125, 250, and 500 μM concentrations. Then, inhibitory pattern analysis against AChE could be determined using a Lineweaver–Burk plot.

Statistical Analysis

All data were expressed as the mean ± standard deviation (SD), analysis of variance was performed for procedures, and significant differences were determined using Duncan’s multiple-range test (p < 0.05) from SAS software (ver. 9.1; SAS Institute, USA).

Antioxidant Effect

Antioxidant activities were evaluated using the ABTS radical scavenging activity and MDA-inhibitory effect assays (Fig. 1). As shown in Fig. 1A, the ABTS radical scavenging activity of FEC was exhibited in a concentration-dependent manner, although the scavenging activity of FEC was lower than for vitamin C used as a positive control at the same concentration. FEC showed 50% scavenging activity (IC₅₀) at 1 mg/ml concentration (Fig. 1B).

Figure 1. Antioxidant activity of fucoidan extract from Ecklonia cava on ABTS radical scavenging activity (A), simple linear regression equation and R-square of ABTS assay (B), inhibitory effect of lipid peroxidation (c) and simple linear regression equation and Rsquare of MDA assay (D). The results are shown as the means ± SD (n = 3), and were statistically considered at p < 0.05. Different small letters indicate a statistical difference.

The inhibition of lipid peroxidation was evaluated by investigating the MDA-inhibitory effect of FEC on both ferric ion- and vitamin C-induced lipid peroxidation from mice brain homogenates (Fig. 1C). FEC showed a greater than 50% MDA-inhibitory effect at a >0.5 mg/ml concentration, and IC₅₀ was shown at 0.40 mg/ml (Fig. 1D).

Neuronal Cell-Protective Effect

The intracellular ROS contents were experimented using the DCF-DA method, and the results in PC-12 cells are shown in Fig. 2A. The H₂O₂-treated group was a negative control (121.31%) and showed significantly increased DCF formation compared with the control group (100.00%), and the vitamin C (81.47%) group was a positive control and showed remarkably decreased intracellular ROS content in the H₂O₂-treated PC-12 cells. However, the FEC groups showed statistically decreased intracellular ROS contents at greater than 20 μg/ml concentrations (102.37%) and the neuronal cell-protective effect of FEC was measured by cell viability against H₂O₂-induced neuronal oxidative damage. The cell viability was evaluated using the MTT reduction method, and the result is indicated in Fig. 2B. The H₂O₂-treated group (48.27%; an approximately 51.73% decrease) showed significant decreased cell viability compared with the control group (100.00%). The FEC group at the 50 μg/ml concentration (85.26%) indicated similar results to the vitamin C (84.21%) groups as a positive control, and showed similar cell viability to the control group at the 100 μg/ml concentration (97.29%).

Figure 2. Neuronal cell-protective effect of fucoidan extract from Ecklonia cava on intracellular ROS contents (A) and cell viability (B) in PC-12 cells, and intracellular ROS contents (C) and cell viability (D) in MC-IXC cells after H₂O₂-induced oxidative damage. The results are shown as the mean ± SD (n = 5), and were statistically considered at p < 0.05. Different small letters indicate a statistical difference.

For MC-IXC cells, intracellular ROS contents are shown in Fig. 2C. The H₂O₂-treated group (141.02%) showed increased DCF formation compared with the control group (100.00%), and vitamin C (77.51%) as a positive control showed decreased intracellular ROS content against H₂O₂ treatment. Meanwhile, the FEC groups showed significantly decreased intracellular ROS contents in human neuroblastoma cells at 20–100 μg/ml concentrations (average 93.90%). The resultant MC-IXC cell viability is indicated in Fig. 2D. The H₂O₂-treated group (81.43%) showed decreased cell viability compared with the control group (100.00%) and the FEC groups showed remarkable cell viability at 50 and 100 μg/ml concentrations. The FEC groups (115.45% at 50 μg/ml and 119.43% at 100 μg/ml concentrations) indicated similar results to that of the vitamin C (116.90%) positive control group, and showed greater cell viability than the control group.

Mitochondrial Activity

The mitochondrial activities of neuronal PC-12 cells were evaluated using the (ΔΨm), ATP levels, and expression levels of mitochondrial-mediated proteins (Fig. 3). The MMP was determined by using the JC-1 dye, and the results are shown in Fig. 3A. The H₂O₂-treated group (60.91%; about 39.09% decrease) showed a significantly decreased JC-1 fluorescence intensity ratio compared with the control group (100.00%), and vitamin C as a positive control showed a slightly higher MMP than that of the H₂O₂-treated group. On the other hand, the FEC groups showed significantly restored MMP against H₂O₂-induced mitochondrial damage at 50 and 100 μg/ml concentrations (100.36% and 110.85%, respectively).

Figure 3. Mitochondrial activity assessments of fucoidan extract from Ecklonia cava. Mitochondrial membrane potential (A), ATP levels (B), band image of western blot analysis (C), and protein expression levels of p-AMPK (D) and BAX (E) from H₂O₂-induced mitochondria damage in PC-12 cells. The results are shown as the mean ± SD (n = 5), and were statistically considered at p < 0.05. Different small letters indicate a statistical difference.

The ATP levels were decreased by H₂O₂ treatment (77.32%; about 22.68% decrease) compared with the control group. Additionally, the ATP levels of the FEC groups were increased by about 1.4 times at 20–100 μg/ml concentrations (average 141.33%) compared with the H₂O₂-treated group.

The mitochondria-mediated protein expression levels of p-AMPK and BAX were also measured, and the results are shown in Figs. 3C-3E. The p-AMPK expression levels were remarkably increased in the H₂O₂-treated group compared with the control group. In contrast, the FEC groups showed significant decreases at 50 and 100 μg/ml concentrations. Furthermore, the BAX protein expression levels were increased by the H₂O₂ treatment. On the other hand, the FEC groups showed decreased BAX protein expression.

Acetylcholinesterase-Inhibitory Effect and Inhibitory Pattern

To evaluate the function of the neurotransmitter in the cholinergic system, we investigated the AChE-inhibitory effect and analyzed the inhibition pattern against AChE (Fig. 4). The inhibitory effect of FEC against AChE showed more than 50% inhibition at the 2 mg/ml concentration, and the IC₅₀ value was shown at the 1.31 mg/ml concentration (Figs. 4A and 4B). The Lineweaver–Burk plot of FEC indicated that upon an increased concentration of the active component, the K_m value of AChE on the active compounds was increased, and the V_max value was decreased (Fig. 4C). The results indicated that FEC has a mixed-type (competitive and noncompetitive) inhibitory pattern against AChE.

Figure 4. Inhibitory effect of fucoidan extract from Ecklonia cava (FEC) against acetylcholinesterase (AChE) (A), simple linear regression equation and R-square of AChE inhibition assay (B) and Lineweaver-Burk plots (1/V vs. 1 /[S]) for three FEC concentrations (C). The results are shown as the mean ± SD or means (n = 5), and were statistically considered at p < 0.05. Different small letters indicate a statistical difference.

In most organisms, oxidative stress is one of the most common causes of disease and could induce neurodegenerative diseases, leading to neuronal cell damage and apoptosis [2]. Recently, there has been increasing interest in materials derived from marine resources that have antioxidant effects as edible and safe substances. However, their antioxidant effects are still unknown, and only studies on the effects of phenolic compounds have been reported [19]. Antioxidant activity could occur not only from a phenolic compound, but also from other components of polysaccharides, such as fucoidan. Fucoidan, as a component of fibrillar cell walls in brown seaweeds, consists of fucose-containing sulfated polysaccharides [20]. In this study, we evaluated potential applications in the food industry by investigating the neuronal cell-protective effect of fucoidan extract from E. cava, which is manufactured to be well digested and absorbed, based on its antioxidant activity.

Above all, we evaluated the in vitro antioxidant activity, and FEC showed ABTS radical scavenging activity and a remarkable lipid peroxidation-inhibitory effect (Fig. 1). The brain tissue is sensitive to oxidative stress due to the high concentrations of polyunsaturated fatty acids and having relatively low activity for antioxidant enzymes, such as glutathione peroxidase and catalase [2]. Therefore, radical scavenging activity and the lipid peroxidation-inhibitory effect may be important for the prevention of neuro-degenerative diseases. Among various polysaccharides extracted from brown and red seaweed, fucoidan and lambda carrageenan have been reported to exhibit the highest free radical scavenging and antioxidant activities [21]. Additionally, in four polysaccharide fractions from brown alga Fucus vesiculosus water extract, fraction 3, containing the fucoidan, exhibited higher antioxidant potential than that of the other fractions containing high alginates, laminarin, and polyphenols [19]. These high antioxidant activities were presumed to be due to their structure containing sulfate contents. In a recent study, a low-molecular-weight fucoidan fraction extracted from Laminaria japonica and its three fractions (DF1, DF2, and DF3) indicated different antioxidant activities. Although DF2 and DF3 had more sulfate contents, DF1 indicated the highest antioxidant activity. Their study demonstrated that the molar ratio of sulfate content to fucose could influence the antioxidant activity rather than sulfate contents [1]. Based on these studies, it can be assumed that the antioxidant effect of FEC is due to the influence of structural features.

According to a previous report, fucoidan derived from E. cava could act as a strong inhibitor against ROS and NO in the LPS-induced inflammatory zebrafish model [22]. Furthermore, a previous study reported that fucoidan from L. japonica effectively attenuated oxidative damage by activating antioxidant enzymes (superoxide dismutase, glutathione peroxidase), decreasing the MDA level and LDH release of H₂O₂-induced neurotoxicity in PC-12 cells. Their study also demonstrated that fucoidan played a key role in anti-apoptosis by activating the phosphoinositide 3- kinase/Akt signaling pathway, which is related to cell growth, proliferation, and differentiation, after exposure to H₂O₂ [23]. In our results (Fig. 2), FEC showed neuronal cellprotective effects on H₂O₂-induced oxidative damage in neuronal cells (PC-12 and MC-IXC cells) by decreasing the intracellular ROS contents and ameliorating the cell viability.

Mitochondrial dysfunctions in AD are considered a major pharmacological target for its therapy [24]. The mitochondrion hypothesis for AD pathogenesis suggests that mitochondrial dysfunction by DNA mutations and oxidative stress are the major causes of AD [24]. Therefore, to evaluate the restorative effect of the mitochondria, we investigated the MMP, ATP level, and p-AMPK protein expression levels as a function of mitochondrial energy metabolism against H₂O₂-oxidative damage in PC-12 cells.

Aβ-induced oxidative stress causes energy metabolism impairment through decreased respiratory enzyme activity, abnormal ROS generation, and disruption of the MMP [2, 5, 24]. The MMP plays an essential role in energy metabolism and free radical generation by converting ADP to ATP via oxidative phosphorylation of the inner membrane [25, 26]. As a result of the reduced MMP, the insufficient ATP levels cause cell apoptosis [27]. ATP is necessary for maintaining cell homeostasis because its hydrolysis is used for synthetic reactions, material transport through membranes, and other endogenous processes [28]. Additionally, the dropped MMP releases cytochrome c from the mitochondria to the cytosol, inducing caspase activation by generating an apoptosome in the presence of apoptotic protease-activating factor 1 [26, 28]. In the FEC-treated PC-12 cell, the increased ATP levels might have been caused by restoring MMP from damage after exposure to H₂O₂ (Fig. 3).

The energy metabolism of the mitochondria is closely related to AMPK activity by maintaining the energetic balance [25]. In abnormal high-oxidative-stress conditions, H₂O₂ inhibits Akt, which is related to cell growth and proliferation, and activates AMPKα as a negative regulator owing to an increase in the AMP/ATP ratio for ATP production in neuronal cells [24]. AMPK may be considered as an important factor for pro-survival kinase, which produces ATP as an energy source by activating the catabolic pathway [29]. However, high oxidative stressinduced prolonged AMPK activation directly elicits the transcriptional induction of Bcl-2 homology domain 3-only protein Bim as a proapoptotic protein, and triggers excitotoxic apoptosis in neurons [30]. In addition, prolonged AMPK activation promotes BAX translocation from the cytosol to the mitochondria via p38 mitogen-activated protein kinase and c-Jun N-terminal kinases pathway activation [29]. Bcl-2 family members, such as BAX and Bcl-2, which have been involved in the apoptosis, were induced by ROS-generating agents [31]. In particular, a proapoptotic protein such as BAX disrupts the MMP and opens the mitochondrial permeability transition pore, and elicits cell apoptosis by activating the caspase pathway [32]. The expression of BAX and p-AMPK proteins were statistically decreased by the FEC treatment in this study (Fig. 3). Based on these reports, FEC might attenuate neuronal cell damage by improving the mitochondrial energy metabolism through elevated ATP levels, improved MMP, and downregulating BAX and p-AMPK against H₂O₂-induced oxidative damage.

The cholinergic system, as a representative role of neuro-transmissional function, is important to neurodegenerative diseases, such as AD in the brain. In particular, cholinergic neurotransmission could be enhanced by AChE inhibition, which increases the acetylcholine (ACh) content as a neurotransmitter in the synapses of neurons [33]. ACh is formed by choline combined with acetyl-CoA, which is synthesized from mitochondrial pyruvate within cholinergic neurons [34]. Therefore, mitochondrial dysfunction inhibits ACh synthesis and decreases neurotransmission, and the AChE inhibitor as a classical pharmacological target of AD therapy is thought to be beneficial by improving the neuronal transmission of ACh. As a result of measuring the AChE-inhibitory activity of 20 kinds of seaweed, E. cava (35.85% at 1mg/ml), Ecklonia kurome, and Myelophycus simplex extracts were reported as the most effective [35]. Compared with these results, FEC seems to have a similar inhibitory effect to E. cava extract of that study, but is slightly lower than tacrine as a positive control. Additionally, FEC showed a mixed (competitive and noncompetitive) inhibitory pattern that was different from that of tacrine (noncompetitive inhibition pattern) (data not shown). The mixed (competitive and noncompetitive) inhibitory pattern is known to bind directly to an enzyme or to an enzyme–substrate complex to more effectively inhibit the action of the enzyme. Therefore, FEC was able to effectively inhibit AChE by binding at both the anionic site inside the enzyme gorge and the peripheral anionic site [36]. In some studies, fucoidan from L. japonica ameliorated learning and memory impairment in Aβ-induced AD rats by improving the oxidative defense system, such as superoxide dismutase and glutathione peroxidase, and regulating the cholinergic system, such as choline acetyl transferase and AChE [37].

Recently reported studies have suggested various physiological activity effects depending on the molecular weight of fucoidan [38-41]. The lowest molecular-weight fucoidan (28.2 kDa) of several sulfated heteropolysaccharides (151.7, 64.5, 58.0, and 28.2 kDa) extracted from Ulva pertusa Kjellm showed the strongest superoxide and hydroxyl radical scavenging activities, reducing power, and metal chelating ability [38]. The absorption mechanism of fucoidan through the gastrointestinal tract is unclear, but the report suggested that fucoidan would be absorbed through the primary intestinal cells [39]. Furthermore, the administration of low-molecular-weight fucoidan (7.6 kDa) indicated a much better absorption rate and bioavailability in plasma and urine than medium-molecular-weight fucoidan (35 kDa), and antithrombotic function can be improved by lowering the molecular weight in a rat model [40]. In addition, fucoidan containing a medium or high molecular weight can act as a prebiotic to improve intestinal health, due to the easily fermentable materials by the intestinal microbiota, and can improve the growth of Lactobacillus strains in the human intestine [41]. In addition, prebiotics have a direct or indirect effect on signaling molecules by regulating gut hormones (e.g., peptide YY as a neuropeptide), hippocampal brain-derived neurotrophic factor, and N-methyl-D-aspartate receptor subunit 1 [41]. Therefore, in future studies, it is necessary to conduct structure and component analyses of the active materials of FEC and the absorption rate and physiological activity in an in vivo model.

In conclusion, fucoidan extract from E. cava showed significant in vitro antioxidant activity, inhibitory effect against AChE, and neuronal cell-protective effect through regulation of the mitochondrial-mediated proteins (p-AMPK and BAX) on H₂O₂-induced neuronal damage. Therefore, FEC with its water-soluble and heat-stable properties could be a good source of natural antioxidants with potential applications for preventing neurodegenerative diseases from oxidative stress-induced neuronal damage, by regulating mitochondrion-mediated proteins.

This research was part of a project titled “Development of Global Senior-friendly Health Functional Food Materials from Marine Resources” funded by the Ministry of Oceans and Fisheries, Korea. We would like to express our deepest gratitude to Seojin Biotech Co. Ltd. for supplying the E. cava fucoidan extract used in this manuscript. Seon Kyeong Park, Jin Yong Kang, Jong Min Kim, Sang Hyun Park, and Bong Seok Kwon were supported by the BK₂1 Plus Program, Ministry of Education, Republic of Korea.

The authors have no financial conflicts of interest to declare.