Preparation of Low-Molecular-Weight Fucoidan by Irradiation-Induced Degradation and Its Protective Effect Against H2O2-Induced Oxidative Stress in RAW 264.7 Cells

TL;DR

Low-molecular-weight fucoidan, made using irradiation, shows strong antioxidant effects in cells and could be a useful dietary supplement.

Contribution

A new method for preparing low-molecular-weight fucoidan using irradiation and its antioxidant efficacy in oxidative stress models is presented.

Findings

Low-molecular-weight fucoidan significantly increased antioxidant enzyme activities and reduced oxidative stress markers in RAW264.7 cells.

The lowest molecular weight fraction (AIF4) showed the highest antioxidant activity and upregulated key antioxidant genes and signaling pathways.

LMWF treatment modulated the PI3K/Akt–Nrf2 signaling axis, suggesting a mechanism for its protective effects against oxidative stress.

Abstract

The biological activities of fucoidan from brown algae have attracted considerable attention. Degradation to low-molecular-weight fucoidan reduces viscosity and improves bioavailability, enhancing antioxidant and anti-inflammatory effects. Fucoidan was degraded using acetic acid combined with 60Co γ-ray irradiation and fractionated by Bio-Gel P10 chromatography to obtain four fractions (AIF1–AIF4). The fractions were structurally characterized and assessed for in vitro radical-scavenging activity and modulation of oxidative stress markers in H2O2-induced RAW264.7 macrophages. Compared with the model group, all fractions significantly increased catalase (CAT) and superoxide dismutase (SOD) activities, with the highest increase of approximately 1.33 U/mgprot for CAT and 20.32 U/mgprot for SOD, while decreasing malondialdehyde (MDA) and intracellular reactive oxygen species (ROS) levels. Among the four fractions, AIF4, with the lowest molecular weight, exhibited the highest antioxidant activity. LMWF treatment also upregulated the mRNA expression of antioxidant-related genes (HO-1, SOD1, SOD2) and signaling molecules (PI3K and Akt), accompanied by increased protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and PI3K/Akt pathway components. These findings indicate that LMWF is closely associated with the regulation of the PI3K/Akt–Nrf2 signaling axis under oxidative conditions and support its potential application as a dietary antioxidant ingredient.

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1. Introduction

Fucoidan primarily consists of sulfated L-fucose, with additional monosaccharide components including galactose, mannose, xylose, arabinose, glucose, and rhamnose. This polysaccharide is predominantly found in brown algae and other marine organisms, including kelp, Sargassum, and sea cucumbers [1]. This macromolecule demonstrates a variety of significant biological activities, including antioxidant, antitumor, anti-inflammatory, and lipid-lowering effects [2]. The biological activity of fucoidan is closely associated with its molecular structure, including molecular weight, types of glycosidic linkages, side-chain architecture, and functional groups. However, high-molecular-weight fucoidan typically exhibits several limitations, such as high solution viscosity, poor solubility, low bioavailability, and limited intestinal absorption by the body, which restrict its practical applications. In contrast, LMWF is more readily absorbed due to its smaller molecular size and often displays enhanced biological activity, thereby exhibiting broader application potential in fields such as pharmaceuticals and functional foods. Consequently, the degradation and structural modification of fucoidan have attracted increasing research interest among scientists worldwide in recent years.

Various methods have been used to degrade fucoidan, including enzymatic hydrolysis, acid hydrolysis, ultrasonic treatment, and irradiation [3]. However, conventional approaches, such as enzymatic and acid hydrolysis, used to obtain LMWF suffer from several limitations, including narrow substrate specificity and reaction conditions that are unfavorable for side-chain cleavage. Moreover, these methods may involve the use of chemical reagents that can disrupt sulfate groups. Irradiation technology is widely applied as an effective approach for material modification and degradation, including electron beam irradiation, γ-ray irradiation, microwave irradiation, and ultraviolet irradiation. Among these techniques, γ-ray irradiation degradation is considered a relatively clean and scalable processing approach, as it can achieve rapid and controllable depolymerization with minimal chemical inputs. Moreover, this method does not require the addition of chemical reagents, rendering it environmentally benign, and under certain conditions, the resulting degradation products can be directly applied without further purification. Therefore, γ-ray irradiation is considered a degradation method with considerable application potential [4,5,6]. This approach offers several practical advantages, including operational simplicity, high degradation efficiency, short processing time, good process controllability, scalability, and the absence of chemical additives. In addition, the degradation products can often be used directly without extensive purification, underscoring the potential of γ-irradiation as a sustainable processing strategy. Cobalt-60 (^60^Co) has a moderate half-life and emits high-energy γ-rays and is therefore widely used as a γ-ray source for irradiation-based degradation [7,8].

γ-Irradiation has been widely applied to polysaccharide degradation. Ni et al. [9] reported that low-dose γ-irradiation promoted moderate depolymerization of high-molecular-weight polysaccharides, thereby improving bioactivity. Similarly, Hojjati et al. [10] observed that γ-irradiation of hawthorn seed polysaccharides significantly enhanced antioxidant capacity and prebiotic potential. Yin et al. [11] further demonstrated that γ-irradiation altered the physicochemical properties of lentinan, thereby influencing its antioxidant, hypoglycemic, and prebiotic functions. Collectively, these studies highlight γ-irradiation as a promising approach for tailoring polysaccharide functionality, thereby supporting applications in the pharmaceutical and functional food industries.

Oxidative stress and the resulting damage are one of the important causes of suboptimal health conditions, and preventing the occurrence of oxidative stress as well as exerting antioxidant effects are the basis for functional food ingredients to perform their functions. Studies have indicated that the antioxidant activity of low-molecular-weight polysaccharides is significantly improved after degradation. Liu et al. [12] obtained a novel jujube polysaccharide (DPZMP3) by degrading polysaccharides. Through research and analysis, it may also be useful for DZMP to enhance its antioxidant activity in vitro. Zhang et al. [13] degraded Sanghuang polysaccharides using β-1,3-glucanase, and the antioxidant activity was significantly enhanced.

In this study, LMWF fractions with different molecular weights were prepared via ^60^Co γ-irradiation. To facilitate controlled depolymerization, fucoidan was pretreated with mild acetic acid prior to ^60^Co γ-irradiation. This pretreatment improves solubility and reduces viscosity, thereby promoting efficient irradiation-induced cleavage while helping retain sulfate-related structural features. The resulting fractions were subsequently separated and purified, followed by detailed structural characterization. Antioxidant activity was systematically evaluated by assessing in vitro radical-scavenging activity as well as protective effects against H_2_O_2_-induced oxidative stress in RAW 264.7 macrophages. This study aimed to provide a theoretical foundation for the further development and application of LMWF as a functional food ingredient. Figure 1 illustrates the overall experimental workflow of this study.

2. Materials and Methods

2.1. Materials

The fucoidan raw material derived from Kjellmaniella crassifolia was provided by the National R&D Branch Center for Seaweed Processing. The fucoidan was extracted from Kjellmaniella crassifolia harvested from the coastal waters of Lüshun, Dalian, China. Its purity was determined to be 95.23%, calculated as the sum of total sugar and sulfate group contents.

The murine RAW 264.7 macrophage cell line was obtained from the Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai, China). Premium-grade fetal bovine serum was obtained from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) Dulbecco’s Modified Eagle Medium (DMEM), methyl thiazolyl tetrazolium (MTT), cell culture–grade dimethyl sulfoxide (DMSO), protein standards, and penicillin–streptomycin solution were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China).

Assay kits for the determination of ROS, SOD, CAT, and MDA were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The universal RNA extraction kit, SYBR Green Pro Taq HS premixed qPCR reagent kit, and Evo M-MLV reverse transcription premix were obtained from Accurate Biotechnology(Hunan) Co., Ltd. (Changsha, China). Ascorbic acid was purchased from Tianjin Bodi Chemical Co., Ltd. (Tianjin, China). All chemicals and reagents used in this study were of analytical grade.

2.2. Preparation of LMWF

Fucoidan (10 g) was dissolved in 500 mL of 0.1 M acetic acid and allowed to equilibrate overnight at room temperature [14]. The solution was then subjected to ^60^Co γ-ray irradiation under ambient conditions (23 ± 2 °C). The irradiation was performed at a total dose of 50 kGy with a dose rate of 2 kGy/h. The irradiation was carried out with the technical support of the Institute for Farm Products Processing and Nuclear-Agricultural Technology, Hubei Academy of Agricultural Science. After irradiation, the sample was concentrated by rotary evaporation and subsequently lyophilized to yield the acetic acid soluble degradation product, designated as AIF-50kGy. To neutralize the acetic acid in AIF-50kGy, 0.1 M NaOH solution was added to adjust the pH to 7. The mixture was then diluted with an equal volume of deionized water. The fucoidan solution was subjected to reverse osmosis to remove sodium acetate. Finally, the solution was concentrated by rotary evaporation and lyophilized to obtain the powder, which was stored for further use.

The LMWF fraction AIF-50kGy (0.1 g) was dissolved in 2 M ammonium bicarbonate solution and fractionated using a Bio-Gel P10 column equilibrated with the same buffer. Elution was performed isocratically at a flow rate of 0.13 mL/min, and fractions containing fucoidan-derived oligosaccharides were collected. The total carbohydrate content of each fraction was quantified using the phenol–sulfuric acid method, and the elution profile was constructed based on absorbance. Fractions enriched in low-molecular-weight components were pooled according to the elution profile, concentrated by rotary evaporation, and subsequently lyophilized to obtain four purified oligosaccharide fractions, designated as AIF1, AIF2, AIF3, and AIF4.

2.3. Free Radical Scavenging Capacity In Vitro

2.3.1. DPPH Radical Scavenging Ability

AIF solutions at different concentrations and a vitamin C (VC) control solution (100 μL) were added to a 96-well plate. Subsequently, 100 μL of 0.1 mol/mL DPPH solution was added. Anhydrous ethanol was used as the blank control. The plate was placed in a microplate reader, shaken for 1 min, and then incubated in the dark for 30 min. The absorbance (OD) was measured at 517 nm [15].

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2.3.2. ABTS+ Radical Scavenging Ability

AIF solutions at different concentrations and a vitamin C (VC) control solution (200 μL) were added to each well. Subsequently, 800 μL of ABTS^+^ working solution was added, mixed thoroughly, and allowed to stand for 6 min. The absorbance (OD) was measured at 734 nm and recorded as A_1_. For the blank control (A_0_), 200 μL of anhydrous ethanol was mixed with 800 μL of ABTS^+^ working solution and processed as described above; the absorbance (OD) at 734 nm was recorded as A_0_ [16].

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2.3.3. OH Radical Scavenging Ability

AIF solutions at different concentrations and a vitamin C (VC) control solution were prepared. For A_1_, FeSO_4_ solution, salicylic acid in ethanol, and H_2_O_2_ solution were added to each sample, and the mixtures were incubated in a 37 °C water bath for 30 min with gentle shaking. The absorbance (OD) at 510 nm was measured and recorded as A_1_. For A_2_, each sample was mixed with 500 μL of 9 mmol/L FeSO_4_, 500 μL of 9 mmol/L salicylic acid in ethanol, and 500 μL of distilled water. After incubation in a 37 °C water bath for 30 min, the mixtures were mixed thoroughly, and the absorbance (OD) at 510 nm was measured and recorded as A_2_. For the blank control (A_0_), 500 μL of distilled water was mixed with 500 μL of 9 mmol/L FeSO_4_, 500 μL of 9 mmol/L salicylic acid in ethanol, and 500 μL of 8.8 mmol/L H_2_O_2_. The mixture was incubated in a 37 °C water bath for 30 min, mixed thoroughly, and the absorbance (OD) at 510 nm was recorded as A_0_ [17].

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2.4. Protective Effects of LMWF on H2O2-Induced Oxidative Stress in RAW 264.7 Cells

2.4.1. Cell Culture

RAW 264.7 cells were cultured in freshly prepared complete medium. The complete medium consisted of DMEM supplemented with fetal bovine serum at a 9:1 (v/v) ratio. Penicillin–streptomycin solution (1 mL per 100 mL of medium) was added to the mixture. Cells were incubated at 37 °C in a humidified incubator with 5% CO_2_.

2.4.2. Establishment of an H2O2-Induced Oxidative Damage Model in RAW 264.7 Cells

Log-phase cells were seeded into 96-well plates at a density of 1 × 10^5^ cells/well, with 200 μL of cell suspension per well. The plates were incubated at 37 °C in a humidified incubator with 5% CO_2_ for 24 h. The culture medium was discarded, and serum-free DMEM containing various concentrations of H_2_O_2_ was added, with eight replicates per group. The plates were further incubated under the same conditions for an additional 24 h. Subsequently, MTT solution was added, and the plates were incubated for 4 h. The culture medium was removed, DMSO (150 μL) was added, and the plates were incubated at 37 °C for 10 min. Absorbance was measured at 490 nm (OD_490_), and cell viability was calculated to determine the optimal H_2_O_2_ concentration.

2.4.3. The Toxic Effects of LMWF on RAW 264.7 Cells

The MTT assay was used to evaluate the cytotoxicity of fucoidan fractions (AIF1–AIF4) in RAW 264.7 cells. Cells were seeded into 96-well plates at 1 × 10^5^ cells/well (200 μL per well) and incubated at 37 °C in a humidified incubator with 5% CO_2_ for 24 h. The culture medium was removed, and 200 μL of complete DMEM containing AIF1, AIF2, AIF3, or AIF4 was added to each well. The final concentrations were 25, 50, 100, 200, 400, 600, 800, and 1600 μg/mL. PBS-treated wells served as the blank control. After incubation for an additional 24 h, 20 μL of MTT solution was added to each well, followed by incubation for 4 h at 37 °C. The culture medium was carefully aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The plates were gently shaken in the dark for 5 min. Absorbance was measured at 490 nm using a microplate reader. Cell viability was calculated, and an appropriate concentration was selected for subsequent experiments.

2.4.4. Determination of Intracellular CAT and SOD Activities and MDA Content

After an additional 24 h of culture as described in Section 2.4.3, the culture medium was carefully aspirated. Serum-free DMEM containing H_2_O_2_ was added to each well, and the cells were incubated for 1 h. Cells were then digested with trypsin and collected into centrifuge tubes using a cell scraper. After centrifugation, the supernatant was discarded, and the cell pellet was washed with PBS, followed by a second centrifugation, after which the supernatant was removed. An appropriate volume of extraction buffer was added according to the manufacturer’s instructions. The cell suspension was sonicated using a pulse mode in an ice-water bath for a total sonication time of 40 min at 100% power, with intermittent cooling to prevent overheating. Subsequently, intracellular CAT and SOD activities, and MDA content, were determined in RAW 264.7 cells according to the manufacturer’s protocols.

2.4.5. Determination of Intracellular ROS Levels

After cells were further cultured for 24 h according to the procedure described in Section 2.4.3, the cells were washed with PBS and the culture medium was removed. An appropriate volume of DCFH-DA, diluted with serum-free DMEM to a final concentration of 10 µM, was added to each well of a six-well plate. The cells were incubated at 37 °C for 20 min in a humidified incubator with 5% CO_2_. Subsequently, the staining solution was removed, and the cells were washed repeatedly and gently resuspended in PBS. Finally, intracellular ROS levels were measured using a fluorescence spectrophotometer.

2.4.6. Effects of LMWF on Oxidative Gene Expression in RAW 264.7 Cells

Cell Preparation

Cells in the logarithmic growth phase were harvested, resuspended, seeded into 6-well plates, and incubated at 37 °C in a humidified atmosphere containing 5% CO_2_ for 24 h. The blank control and VC positive control groups were cultured in serum-free DMEM, whereas the experimental groups were cultured in serum-free DMEM supplemented with AIF1–AIF4. After 24 h, serum-free DMEM containing 300 μmol/L H_2_O_2_ was added, and RNA was extracted after 1 h.

Real-Time Quantitative PCR Reaction

Total RNA was extracted using an RNA extraction kit according to the manufacturer’s instructions, and RNA concentration and purity were determined using a NanoDrop spectrophotometer. Complementary DNA synthesis was then performed using a reverse transcription kit under standard conditions. The messenger RNA expression levels of Nrf2, HO 1, SOD1, SOD2, Akt, and PI3K were quantified using SYBR Green time quantitative PCR, with β-actin serving as the internal reference gene. The primer sequences used for gene amplification are shown in Table 1. Relative gene expression levels were calculated using the 2^−ΔΔCq^ method.

2.4.7. Western Blot Analysis

Protein levels of Nrf2, PI3K, and Akt in RAW 264.7 cells were determined by Western blotting. RAW 264.7 cells were exposed to H_2_O_2_ and then treated with fucoidan oligosaccharide fractions (AIF1–AIF4) for 24 h. Total protein was extracted using RIPA lysis buffer, and nuclear protein was subsequently extracted according to the manufacturer’s instructions. Protein concentrations were quantified. Proteins were separated by SDS–PAGE and transferred onto PVDF membranes, followed by blocking with 5% blocking reagent for 2 h. Membranes were incubated with primary antibodies in TBST containing 5% bovine serum albumin (BSA) overnight at 4 °C. After washing with TBST, membranes were incubated with secondary antibodies at room temperature for 1 h, followed by additional washing with TBST. Protein bands were visualized using enhanced chemiluminescence (ECL) and imaged using a Tanon-5200 chemiluminescent blot detection system.

2.5. Determination of Total Carbohydrates

Total carbohydrate content was determined using the phenol–sulfuric acid method [18]. A standard sugar mixture (L-fucose and D-galactose, 3:1, w/w) was accurately weighed (4 mg, dried to constant weight), dissolved in distilled water, and diluted to a final volume of 100 mL. Aliquots of the standard solution at different concentrations were prepared, diluted with distilled water to a final volume of 2 mL, and then mixed sequentially with 6% (w/v) phenol and concentrated sulfuric acid. After rapid mixing and cooling to room temperature, the absorbance was measured at 490 nm. A standard curve was constructed by plotting absorbance against the mass of total sugar (mg) in each reaction mixture. The total sugar content of unknown samples was determined using the same procedure, and absorbance values were interpolated from the standard curve.

2.6. Determination of Sulfate Content

Sulfate content was quantified using the barium chloride–gelatin method [18]. Potassium sulfate (K_2_SO_4_), dried to constant weight, was dissolved in 1 mol/L hydrochloric acid (HCl) to prepare a standard stock solution. Aliquots of the standard solution at different concentrations were transferred to colorimetric tubes, and 1 mol/L HCl was added; a tube containing only HCl served as the blank control. Trifluoroacetic acid and barium chloride solution were then added sequentially, followed by thorough mixing and incubation for 20 min. The absorbance at 360 nm was measured and recorded as A_1_. Subsequently, 1 mL of 0.5% gelatin solution was added, and the absorbance was measured again under the same conditions to obtain A_2_. A standard curve was generated by plotting the absorbance difference (A_1_ − A_2_) against the corresponding sulfate content. The sulfate content of fucoidan samples was determined using the same procedure, and the measured (A_1_ − A_2_) values were interpolated from the standard curve.

2.7. Determination of Monosaccharide Composition

Pre-column derivatization high-performance liquid chromatography (HPLC) was employed for monosaccharide composition analysis [19]. Fucoidan (10 mg) was weighed into a hydrolysis tube, and 5 mL of 4 mol/L trifluoroacetic acid (TFA) was added. The mixture was hydrolyzed at 110 °C for 8 h in a constant-temperature forced-air oven. After cooling to room temperature, the hydrolysate was gradually neutralized with sodium hydroxide solution, and the final volume was adjusted to 5 mL with deionized water.

Mixed monosaccharide standards (Fuc, Gal, Man, Rha, Xyl, GlcA, and GalA), together with fucoidan samples irradiated at different doses, were transferred to reaction tubes. Solutions of 1-phenyl-3-methyl-5-pyrazolone (PMP) and NaOH were added, and the mixture was incubated at 70 °C for 40 min. After cooling to room temperature, the reaction was terminated by adding 0.3 mol/L HCl. Chloroform was added, the mixtures were vigorously shaken, and phase separation was allowed for 10–20 s. The lower organic phase was carefully removed, and the extraction was repeated 3–5 times. The aqueous phase was filtered through a 0.22 μm organic membrane, and a 20 μL aliquot was injected for HPLC analysis.

Chromatographic separation was performed on a C18 column using gradient elution at a flow rate of 1.0 mL min^−1^. The mobile phases were as follows: A, 15% (v/v) acetonitrile with 0.05 mol/L phosphate buffer (KH_2_PO_4_–NaOH); B, 40% (v/v) acetonitrile with 0.05 mol/L phosphate buffer (KH_2_PO_4_–NaOH, pH 6.80); and C, 5% (v/v) methanol. The gradient program was set with time points at 0, 10, 55, and 65 min.

2.8. FTIR Spectroscopic Analysis

Fourier-transform infrared (FTIR) spectroscopy was performed using the KBr pellet method [20]. Fucoidan samples (2 mg) obtained after irradiation at different doses were mixed with KBr (150 mg) and dried in a forced-air oven at 105 °C to constant weight. The mixtures were ground into a homogeneous powder and pressed into transparent pellets. The FTIR spectra were collected using a Nicolet iS50 FTIR spectrometer (Thermo Nicolet, Waltham, MA, USA) over the range of 4000–400 cm^−1^, with a blank KBr pellet (150 mg) used as the background.

2.9. Determination of Molecular Weight

The relative molecular weight distribution of AIF was determined using high-performance gel permeation chromatography (HPGPC) coupled with a multi-angle laser light scattering (MALS). The experimental conditions were as follows: a flow rate of 0.6 mL/min; an SB HQ 804 column connected in series with an SB HQ 802.5 column; Na_2_SO_4_ solution as the mobile phase; and an injection volume of 100 μL.

Liquid chromatography–mass spectrometry (LC–MS) analysis was performed on purified AIF2, AIF3, and AIF4 fractions. The system comprised an Agilent 1290 ultra-high-performance liquid chromatography (UHPLC) instrument coupled with an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). A Phenomenex Luna HILIC column (150 mm × 5 μm) was used with negative-ion electrospray ionization (ESI). Mobile phase A was an aqueous ammonium acetate solution, and mobile phase B was 98% acetonitrile containing ammonium acetate. The column temperature was 25 °C, the injection volume was 5 μL, and the flow rate was 0.15 mL/min. Data were processed using DeconTools and GlycoRsoft for spectral deconvolution. Fucose oligomers with varying degrees of polymerization and sulfation were identified, and their relative abundances (%) were calculated.

2.10. Data Analysis

Data from three independent replicates for each indicator are presented as mean ± standard deviation (SD), and analysis of variance (ANOVA) was performed using SPSS 21.0 to determine statistical significance (p < 0.05 for significant differences and p < 0.01 for highly significant differences).

3. Results and Discussion

3.1. Preparation of LMWF

AIF-50 kGy, generated by γ-irradiation, was subjected to nanofiltration, rotary evaporation, and freeze-drying. Subsequent purification on a Bio-Gel P10 column yielded four fractions (Figure 2), designated as AIF1–AIF4, with respective yields of 39.64%, 18.57%, 11.95%, and 15.95%.

3.2. Chemical Composition Analysis of LMWF

As shown in Table 2, the total sugar contents of AIF1–AIF4 were higher than that of AIF-50 kGy, indicating that the polysaccharide fractions purified by Bio-Gel P10 exhibited relatively high purity. The sulfate contents of AIF3 and AIF4 were slightly higher than that of AIF-50 kGy, whereas AIF1 and AIF2 exhibited lower sulfate contents. This difference may be attributed to the elution behavior on the Bio-Gel P10 column, where AIF1 and AIF2 eluted earlier and possessed higher molecular weights, while AIF3 and AIF4 eluted later.

Monosaccharide composition is a key determinant of fucoidan structure, and fucoidans typically comprise multiple monosaccharides in different proportions. As shown in Table 2, AIF1 and AIF2 were enriched in glucuronic acid, xylose, and fucose. After further fractionation by Bio-Gel P10, the proportions of mannose, glucuronic acid, galacturonic acid, and xylose decreased in AIF3 and AIF4, whereas fucose became the predominant monosaccharide. The fucose content followed the order AIF4 > AIF3 > AIF2 > AIF1, and AIF4 reached 92.54%, suggesting that AIF4 is a relatively simple, fucose-rich sulfated oligosaccharide. Based on monosaccharide composition analysis, AIF1 was identified as an LMWF oligosaccharide mainly composed of fucose, xylose, and mannose, with a relative molecular mass of 14.06–15.98 kDa. AIF2–AIF4 contained different degrees of sulfate substitution. AIF2 was enriched in fucan trisaccharides, whereas AIF3 and AIF4 were enriched in fucan monosaccharides; all fractions were mixed-type oligosaccharides. AIF4 possessed the lowest relative molecular weight, ranging from 243 to 531 Da. Choi et al. [21] proposed that high-molecular-weight fucoidans are more susceptible to irradiation-induced, radical-mediated cleavage, resulting in more extensive degradation, whereas degradation rates decrease as molecular weight decreases.

3.3. FTIR Spectroscopy of LMWF

In FTIR spectra, several absorption bands are characteristic of polysaccharides. Specifically, the characteristic O–H absorption band appears in the wavenumber range of 3200–3600 cm^−1^, while the characteristic C–H stretching vibration appears in the range of 2800–3000 cm^−1^. Moreover, absorption features in the wavenumber range of 800–1200 cm^−1^ are commonly used to infer the structural characteristics and linkage patterns of polysaccharides.

The FTIR spectra of the LMWF fractions (AIF1–AIF4) are shown in Figure 3. All fractions exhibited a broad and intense band in the range of 3200–3400 cm^−1^, which was attributed to O–H stretching vibrations. The C–H stretching vibration band around 2940 cm^−1^ corresponds to methyl groups in fucoidan, while a characteristic C=O stretching band was also observed near 1600 cm^−1^. The S=O symmetric stretching vibration near 1255 cm^−1^ and the C–O–S stretching vibration near 848 cm^−1^ are characteristic bands of the sulfate ester groups in fucoidan. The C–O–C and C–OH stretching vibrations around 1130 cm^−1^ are characteristic of the polysaccharide backbone. Enhanced absorption near 1255 cm^−1^ was observed for AIF3 and AIF4, and AIF4 exhibited a significantly larger absorption band near 848 cm^−1^ than the other fractions, indicating a higher sulfate content, which is consistent with the sulfate content determination results. Moreover, the absorption peak at approximately 848 cm^−1^ in AIF4 suggests sulfate substitution at the C4 position of fucose residues.

3.4. In Vitro Antioxidant Activity of LMWF

3.4.1. Scavenging Capacity for DPPH, ABTS+, and ·OH Free Radicals

As shown in Figure 4, the radical-scavenging activities of fucoidan against DPPH, ABTS^+^, and ·OH radicals increased with increasing concentration. The DPPH-scavenging activities of AIF1–AIF4 increased in a concentration-dependent manner. At the same concentration, AIF4 consistently exhibited higher scavenging rates than the other fractions, indicating the strongest scavenging capacity against DPPH, ABTS^+^, and ·OH radicals. At 10 mg/mL, the DPPH and ·OH-scavenging rates of AIF4 reached 97.70% and 98.36%, respectively. At a concentration of 10 mg/mL, the scavenging rates of all fractions exceeded 85%. At 10 mg/mL, the ABTS-scavenging rates of all fractions approached that of VC. Collectively, these results indicate that all four fractions exhibit antioxidant activity to varying extents, with AIF4 displaying the highest activity.

3.4.2. Establishment of an H2O2-Induced Oxidative Damage Model in RAW 264.7 Cells

The effect of H_2_O_2_ on the viability of RAW 264.7 macrophages was evaluated using the MTT assay. The results, plotted as a function of H_2_O_2_ concentration, are shown in Figure 5. As the H_2_O_2_ concentration increased, the viability of RAW 264.7 cells gradually decreased, indicating that H_2_O_2_ impaired cell viability. At 250 μmol/L H_2_O_2_, a significant inhibitory effect on cell viability was observed (p < 0.05). At 300 μmol/L H_2_O_2_, cell viability decreased to 61.6% ± 1.46%, indicating a highly significant inhibitory effect. At this concentration, morphological and structural alterations were observed in RAW 264.7 macrophages. Therefore, 300 μmol/L H_2_O_2_ was selected to induce oxidative injury in RAW 264.7 cells for subsequent experiments assessing the protective effects of AIF1–AIF4.

3.4.3. Effects of LWMF on the Viability of RAW 264.7 Cells

Higher cell survival rates indicate enhanced cell viability and proliferation, suggesting lower cytotoxicity. As shown in Figure 6, AIF1–AIF4 exerted concentration-dependent effects on the viability of RAW 264.7 cells. For AIF1, cell viability gradually increased at 25–100 μg/mL but progressively decreased at 200–1600 μg/mL, with inhibitory effects observed at 800 and 1600 μg/mL. AIF2 induced an initial increase followed by a decrease in cell viability, with the strongest promotive effect observed at 200 μg/mL. AIF3 significantly enhanced cell viability at concentrations of 25–400 μg/mL, with the highest viability observed at 400 μg/mL. AIF4 significantly promoted cell viability across all tested concentrations, with the strongest effect observed at 400 μg/mL. Overall, AIF2–AIF4 showed increasing trends in cell viability within the concentration range of 25–200 μg/mL, accompanied by high survival rates. Although AIF1 showed a decreasing trend at 200 μg/mL, it still significantly promoted cell viability compared with NC. In this study, a concentration of 200 μg/mL for each fraction was selected as the dose for subsequent experiments.

3.5. Protective Effects Against H2O2-Induced Oxidative Stress in RAW 264.7 Cells

The activities of CAT and SOD in cells treated with AIF1, AIF2, AIF3, and AIF4 are shown in Figure 7A,B. Compared with the normal control (NC) group, the H_2_O_2_-induced model group exhibited a significant reduction in CAT and SOD activities (p < 0.01). In contrast, pretreatment with vitamin C (VC) or AIF1–AIF4 significantly increased CAT and SOD activities compared with the model group (p < 0.01). Specifically, SOD activity increased by 51.77%, 26.81%, 30.24%, 42.63%, and 45.21% in the VC, AIF1, AIF2, AIF3, and AIF4 groups, respectively, relative to the model group. Among the fucoidan oligosaccharide fractions, AIF3 and AIF4 showed higher CAT and SOD activities than AIF1 and AIF2, with AIF4 exhibiting the strongest effect. Wang et al. [22] investigated the protective effects of polysaccharides (MPA) isolated from Salsola soda against H_2_O_2_-induced damage in RAW 264.7 cells. Consistent with our findings, they reported that MPA protected cells from injury by enhancing CAT activity and maintaining cell viability. Meenakshi et al. [23] evaluated the antioxidant and anti-inflammatory potential of sulfated polysaccharides (PS) isolated from seaweed by monitoring oxidative stress and inflammatory markers in RAW 264.7 cells. PS-treated cells exhibited significantly increased SOD activity, consistent with the present results showing that fucoidan-derived fractions elevated SOD activity in RAW 264.7 cells.

MDA content reflects the extent of intracellular lipid peroxidation and free-radical attack, with higher MDA content indicating more severe cellular damage. As shown in Figure 7C, MDA content in the model group was significantly increased compared to that in the NC (blank) group (p < 0.01). The VC positive control group showed significantly lower MDA content than the blank group (p < 0.05), indicating that VC inhibited MDA accumulation and reduced lipid peroxidation. Compared with the model group, MDA content was significantly reduced in the VC group as well as in AIF-treated groups; no significant differences were observed between the AIF-treated groups and the NC group. Although MDA content in the AIF-treated groups was not significantly lower than that in the NC group, it was significantly lower than that in the model group, indicating that pretreatment with AIF1–AIF4 reduced MDA production and lipid peroxidation. Among the four fractions, AIF4 showed the greatest effect, possibly because of its lowest molecular weight (243.02–389.29 Da), which may facilitate more effective radical scavenging. Xie et al. [24] reported that phosphorylated Cynara scolymus polysaccharide (P-CP) significantly reduced MDA levels and protected RAW 264.7 cells against H_2_O_2_-induced oxidative stress through an integrated antioxidant defense mechanism.

The effects of AIF1–AIF4 on intracellular ROS levels are shown in Figure 7D. Compared with the NC group, H_2_O_2_ stimulation caused a highly significant increase in intracellular ROS levels (p < 0.01), confirming oxidative stress induction in RAW 264.7 cells. Compared with the model group, intracellular ROS levels in the VC positive control group decreased significantly by 54.7% (p < 0.01). Pretreatment with AIF1, AIF2, AIF3, or AIF4 significantly reduced intracellular ROS levels, with AIF4 showing the strongest effect. Collectively, these results demonstrate that the sulfated fucan oligosaccharide fractions (AIF1–AIF4) attenuated H_2_O_2_-induced ROS accumulation in RAW 264.7 cells, thereby exerting antioxidant effects. This observation is consistent with the results reported by Liang et al. [25] on sulfated polysaccharides isolated from Haematococcus pluvialis in RAW 264.7 cells. Because sulfate group content is a key determinant of antioxidant activity, the superior activity of AIF3 and AIF4 is likely attributable, at least in part, to their higher sulfate content.

3.6. The Effect of LMWF on the Expression of Antioxidant-Related Genes in RAW 264.7 Cells

With the relative gene expression levels of the blank group normalized to 1, the mRNA expression levels of Nrf2, SOD1, SOD2, PI3K, and Akt were quantified in H_2_O_2_-induced oxidatively damaged RAW 264.7 cells treated with AIF1–AIF4. The results are shown in Figure 8A,C–F. Compared with the blank group, the H_2_O_2_-induced model group exhibited significantly reduced expression of Nrf2, SOD1, SOD2, PI3K, and Akt (p < 0.01), with relative expression levels of 0.53, 0.60, 0.42, 0.39, and 0.51, respectively. Relative expression levels of these genes in the VC positive control group and AIF1–AIF4 groups were significantly increased (p < 0.01) compared with the model group. Among these treatments, AIF4 exerted the most pronounced effect, with expression levels of 1.21, 0.95, 1.05, 0.93, and 0.88 for Nrf2, SOD1, SOD2, PI3K, and Akt, respectively, exceeding the levels observed in the blank group and indicating a significant ameliorative effect on oxidative damage in RAW 264.7 cells. Fu et al. investigated the protective effect of paclitaxel (RPC) against oxidative damage in RAW 264.7 cells. Using qRT-PCR to quantify SOD1 and SOD2 mRNA expression, they reported that RPC induced a dose-dependent increase in SOD1 and SOD2 mRNA expression in RAW 264.7 cells and effectively reduced the H_2_O_2_-induced cell damage rate.

The effects of AIF1–AIF4 on HO-1 mRNA expression in RAW 264.7 cells are shown in Figure 8B. Compared with the NC group, HO-1 mRNA expression in the model group was significantly decreased (p < 0.01), with a relative expression level of 0.46. Compared with the model group, HO-1 mRNA expression was significantly increased in the VC positive control group and in cells pretreated with AIF1–AIF4, with AIF3 and AIF4 exhibiting more pronounced effects than AIF1 and AIF2. Consistent with these findings, Li et al. [26] reported that tea polyphenols (TPs) significantly increased Nrf2 and HO-1 mRNA expression, thereby protecting cells from H_2_O_2_-induced oxidative damage.

3.7. Effects of LMWF on Nrf2/PI3K/Akt Protein Expression in H2O2-Induced RAW 264.7 Cells

Studies by Galiè et al. [27] indicate that ozone-induced activation of the antioxidant response element (ARE) can be reversed by ectopic expression of Kelch-like ECH-associated protein 1 (Keap1), an inhibitor of Nrf2. The Nrf2 signaling pathway plays a crucial role in the antioxidant response to mild ozone exposure. Under normal physiological conditions, Nrf2 is retained in the cytoplasm by Keap1, which promotes Nrf2 degradation. Upon oxidative stimulation, Nrf2 dissociates from Keap1, translocates to the nucleus, and transactivates ARE-driven genes, thereby inducing the expression of downstream antioxidant enzymes. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway is closely associated with oxidative stress damage processes in multiple organs and tissues, and activation of this pathway contributes to mitigating oxidative injury. Li et al. [28] reported that PI3K/Akt activation is positively correlated with nuclear Nrf2 levels. The PI3K/Akt pathway serves as an upstream regulator of Nrf2, and its activation promotes Nrf2 nuclear translocation, thereby enhancing phase II enzyme expression.

The protein expression levels of Nrf2 and key components of the PI3K/Akt signaling pathway were analyzed by Western blotting to elucidate the antioxidant mechanisms of fucoidan fractions AIF1, AIF2, AIF3, and AIF4 in H_2_O_2_-induced RAW 264.7 cells. The protein expression results are presented in Figure 9. As shown in Figure 9B, compared with the normal control (NC) group, Nrf2 protein expression in the H_2_O_2_-induced model group was significantly reduced (p < 0.01). In contrast, Nrf2 protein expression was significantly increased in the vitamin C (VC) positive control group and in RAW 264.7 cells treated with AIF2, AIF3, and AIF4 (p < 0.01), with the highest expression observed in the AIF4-treated group. As shown in Figure 9C,D, the protein levels of PI3K and Akt in the model (M) group were significantly reduced compared with those in the NC group (p < 0.01). In contrast, treatment with VC as well as AIF3 and AIF4 significantly upregulated PI3K and Akt protein levels compared with the model group (p < 0.01). Notably, PI3K protein levels in cells treated with AIF1 and AIF2 were lower than those in the AIF3- and AIF4-treated groups (p < 0.05 and p < 0.01, respectively). Akt protein expression was significantly increased in the AIF2-treated group (p < 0.05), whereas no significant change in Akt expression was observed in the AIF1-treated group. These results demonstrate that AIF4 exerted the most pronounced effect on Nrf2 and PI3K/Akt pathway–related protein expression among the four fractions. Collectively, AIF1–AIF4 activated the PI3K/Akt-mediated signaling pathway, thereby promoting Nrf2 expression, alleviating H_2_O_2_-induced oxidative damage in RAW 264.7 cells, and ultimately exerting antioxidant effects.

4. Conclusions

This study employed ^60^Co γ-irradiation to degrade fucoidan and generate irradiation products (AIF) at doses ranging from 0 to 50 kGy. Subsequent fractionation using a Bio-Gel P10 column yielded four fucoidan-derived fractions (AIF1–AIF4). Comprehensive analyses of chemical composition, structural characteristics, and in vitro antioxidant activity demonstrated that AIF4 exhibited the strongest antioxidant activity, likely due to its lower molecular weight and higher sulfate and fucose contents. All fractions (AIF1–AIF4) significantly increased CAT and SOD activities in H_2_O_2_-treated RAW 264.7 cells, markedly reduced MDA levels and intracellular ROS levels, and upregulated the expression of multiple genes, including Nrf2. These fractions also increased the protein expression levels of Nrf2 and PI3K in H_2_O_2_-treated RAW 264.7 cells, thereby alleviating oxidative stress. Collectively, these findings suggested that γ-irradiation is an effective approach for fucoidan degradation and may enhance its potential application as a marine-derived functional food or nutraceutical ingredient. Future studies should validate the in vivo efficacy, safety, and bioavailability of AIF4, further verify the PI3K/Akt–Nrf2 mechanism using pathway inhibition or gene silencing, and establish scalable processing and quality-control criteria together with stability in food matrices and under simulated gastrointestinal conditions, thereby supporting the translation of AIF4 into functional food and nutraceutical applications.