Extraction-driven structural modulation of fucoidans from Sargassum assimile enhances antioxidant and immune-stimulating activities

Abstract

Fucoidans are multifunctional marine polysaccharides with promising therapeutic potential, yet their structure–function relationships remain insufficiently understood. This study investigates the structure–function relationship of fucoidans extracted from Sargassum assimile using different extraction conditions and subsequent molecular weight fractionation. Fucoidans were extracted using distilled water and 0.025 M hydrochloric acid (2, 4 and 8 h), followed by ultrafiltration to fractionate polysaccharides (2, 10 and 30 kDa). Acidic extraction significantly increased yield, sulfate content and fucose levels compared to aqueous extraction. Prolonged acid exposure reduced molecular weight and altered conformation. Fractionation demonstrated that lower molecular weight fractions (2–10 kDa) exhibited superior antioxidant activity, while medium fractions (10–30 kDa) enhanced immunostimulatory effects, inducing nitric oxide production in RAW264.7 macrophages. Structural analysis highlighted that sulfate content and molecular weight are important factors influencing these biological properties. Overall, these findings suggest that controlled extraction and fractionation strategies can be applied to tailor the biological functions of fucoidans, thereby paving the way for the sustainable development of high-value functional ingredients from S. assimile within the emerging blue bioeconomy.

Highlights

• •Acidic extraction boosts fucoidan yield (6.9 %) and sulfate content (17.4 %).
• •Low-MW fucoidans (2–10 kDa) show highest antioxidant activity (61.19 %).
• •Medium-MW fucoidans (10–30 kDa) enhance macrophage NO production (26.54 μM).
• •Controlled HCl extraction reduces MW while preserving bioactivity.
• •Ultrafiltration enables targeted fractionation by molecular weight.

1. Introduction

Seaweeds are the essential components of aquatic ecosystems that have been the subject of scientific studies for years due to their significant role in marine life (Cotas et al., 2023). Over the course of the past few decades, the identification of the extensive structural complexity of seaweed metabolites has revolutionized the pharmaceutical and food industries (Carpena et al., 2024). The consumption of seaweeds has been associated with a range of health-promoting effects such as anticancer, antioxidant, antiviral, anti-inflammatory, antihypertensive and anticoagulant properties (Carpena et al., 2024; Jegadeshwari & Rajaram, 2024). The aforementioned therapeutic functions basically stem from the abundance of polysaccharides, vitamins, minerals, polyunsaturated fatty acids (PUFA), phenolic compounds, carotenoids and proteins (Mirza et al., 2024). Polysaccharides, such as fucoidan, laminarin, carrageenan, ulvan, agar and alginate constitute an essential part of the seaweed structure (Jegadeshwari & Rajaram, 2024). Fucoidans have been used as bioactive ingredients to prevent and treat a wide range of human diseases because they are known to have a wide range of therapeutic effects and low toxicity (Sarkar et al., 2024). Fucoidan is a type of polysaccharide that is found in the fibrillar cell walls and intercellular spaces of brown seaweeds and contains fucose as the major sugar (Chaloshtori et al., 2023). These fucose-containing sulfated polysaccharides are interconnected inside a complex matrix of alginates, proteins and low molecular weight substances via ionic and hydrogen interactions (Skriptsova, 2015). According to Cumashi et al. (2007), fucoidans are made up of a chain of L-fucose with sulfate groups attached at C-2 or C-4 positions and a polymer chain of (1 → 3)-α-L-fucopyranose or/both (1 → 3)- and (1 → 4)-α-L-fucopyranose residues. Fucoidans are classified as a group of heteropolysaccharides meaning that, in addition to fucose, they contain varying contents of other sugars in their chain structure including galactose, mannose, glucose, xylose, rhamnose and arabinose (Bahramzadeh et al., 2019; Tabarsa et al., 2020). Considering the close correlation between chemical structure and biological effects of fucoidans, it is known that various factors influence their expected properties, with the most significant being variations in their molecular and chemical structures primarily due to intrinsic factors such as species variation, seaweed cultivation conditions and isolation techniques (Sánchez-Camargo et al., 2016). Molecular weight, sulfate quantity and positioning, the presence of additional functional groups, monosaccharide composition, type of glycosidic linkages and molecular conformation are the major structural contributors that ultimately govern the biological properties of fucoidans (Kim et al., 2015; Senthil, 2024). Here, the molecular weight of seaweed polysaccharides plays a significant role in their biological properties, especially the activation of immune cells. Fucoidans have a diverse spectrum of molecular weights ranging from 1 to 52 × 10⁵ g/mol in which low molecular weight polysaccharides demonstrate increased immunostimulatory function (Borazjani et al., 2017; Park et al., 2024; Zhao et al., 2016). According to Wang et al. (2023), the biological efficacy of fucoidans with high molecular weights is believed to be restricted due to their weak solubility, high viscosity and considerably large dimensions, which inhibit the expression of a full-scale therapeutic effect. So far, various studies have been conducted on fucoidans to produce low molecular weight molecules with enhanced bioactivities, tackling these major challenges (Zayed et al., 2020). Multiple degradation approaches, including the use of specific enzymes (Kusaykin et al., 2008), mineral and organic acids (Mousavi et al., 2020) and free radicals (He et al., 2017) in association with physical means such as ultrasound (Torres et al., 2018) and microwave (Balboa et al., 2013), are introduced to either cleave the glycosidic linkages thereby producing low-molecular weight fucoidans or break the hydrogen bonds forming aggregates in solutions that can hide the behavior of individual polysaccharide chains. However, the biological activities of low molecular weight fucoidans may be compromised during the hydrolysis process due to secondary structural alterations, including the levels of functional groups and sugar content (Suprunchuk, 2019). Besides, the implementation of a separate modification process imposes additional economic costs and environmental concerns. Therefore, finding a balance between maintaining or enhancing the biological activities of fucoidans and minimizing the shortcomings associated with their production is crucial for academic investigations and commercial applications. One potential solution could involve optimizing the extraction process using acids as the most conventional and economically feasible methods to produce fucoidans with distinctively different chain length and molecular conformation. Additionally, an alternative downstream processing step to costly and lengthy chromatographic purifications could be the utilization of cross flow ultrafiltration, which allows for efficient separation of fucoidans based on their molecular sizes (Zayed & Ulber, 2019; Ramírez-Partida et al., 2024). Therefore, in order to obtain fucoidans with different molecular characteristics, this research implemented comparative extraction process using water and acids with different concentrations, which was subsequently followed by an ultrafiltration procedure to fractionate the polysaccharides based on their sizes. This study uniquely compares water and acid extraction methods followed by ultrafiltration to produce fucoidans of distinct molecular sizes, directly linking extraction conditions to nitric oxide–mediated immunostimulatory activity. It provides new insights into optimizing acid extraction as a superior, economical alternative to conventional purification techniques for bioactive fucoidan production. Such studies are essential for establishing structure–function relationships that guide the sustainable production of marine-derived biopolymers with targeted bioactivities. They provide a scientific foundation for developing value-added applications of seaweed polysaccharides in health, food and pharmaceutical sectors, while promoting the efficient and environmentally responsible use of marine resources. These findings hold potential for promoting health-oriented food products and pharmaceutical formulations, fostering a circular blue bioeconomy and generating socio-economic benefits through the sustainable valorization of marine biomass.

2. Materials and methods

2.1. Seaweed preparation

Fresh specimens of Sargassum assimile were sampled from the Chabahar coast, Iran during winter. The seaweed was carefully hand-picked to avoid collecting any marine debris or other organisms. The collected samples were immediately rinsed with seawater to remove any adhering sand, debris or salt. Subsequently, the seaweed was washed thoroughly with fresh water until the water ran clear. The cleaned seaweed was then spread in a single layer on trays and dried in oven at 60 °C for until completely dehydrated. The dried seaweed was subsequently ground into a fine powder and sieved through a 0.5-mm sieve to ensure uniform particle size. The sieved powder was packaged in plastic bags and stored at −20 °C for further analysis.

2.2. Depigmentation and extraction of fucoidans

Initially, 50 g of milled samples were subjected to treatment with 95 % ethanol while being mechanically stirred for 2 h to separate unfavorable compounds and pigments (Fig. 1A). The solid phase was isolated from the liquid phase using centrifuge (8000 rpm, 10 °C, 10 min), and this procedure was repeated until the ethanol turned colorless. Following a single acetone wash, the samples were dried and stored at room temperature for a 24 h. For the aqueous extraction of fucoidans, 10 g of seaweed samples were immersed in distilled water at 60 °C and the supernatant was harvested and concentrated after centrifugation (8000 rpm, 25 °C, 10 min). Subsequently, to eliminate alginate from fucoidan, 1 % calcium chloride was introduced to the supernatant, facilitating the removal of the precipitated calcium alginate. Ultimately, the extracted fucoidans were recovered using 70 % ethanol, washed with acetone and dried at ambient temperature. For the acid extraction of fucoidans, 10 g of seaweed samples were immersed in 0.025 M hydrochloric acid for durations of 2, 4 and 8 h at 60 °C to yield polysaccharides. Subsequently, the supernatant was obtained by centrifugation (8000 rpm, 10 °C, 10 min) and the acidic extract was neutralized using 0.05 sodium hydroxide. Subsequently, following the elimination of alginic acid from the solution, fucoidans were recovered using 70 % ethanol, washed with acetone and dried at ambient temperature (Chaloshtori et al., 2023).

Fig. 1.

Schematic workflow (A) for fucoidan extraction from S. assimile. Extraction conditions included aqueous (AS-D2: 60 °C, 2 h) and acidic (AS-A2, AS-A4, AS-A8: 0.025 M HCl, 2–8 h, 60 °C), followed by a Vivaflow 200 cross-flow ultrafiltration system (2, 10 and 30 kDa). Visual appearance (B) and colorimetric analysis (C-E) (L*, a*, b*) of fucoidans extracted under different conditions. Color attributes were L* for lightness, a* for redness and b* for yellowness. A microspectrophotometer (model 3/18, Sheen Instruments, UK) was to analyze the color attributes. Superscript small letters indicate significant differences between fucoidans (p < 0.05).

2.3. Cross-flow ultrafiltration

In order to fractionate the fucoidans, an ultrafiltration system was used to separate polysaccharide samples based on particle size. Initially, 200 mg of fucoidan was dissolved in distilled water at a temperature of 60 °C and fractionation performed using a cross-flow ultrafiltration system with molecular weight (M_w) cut-offs of 2, 10 and 30 kDa (Vivaflow 200). Finally, the fucoidan fractions were freeze-dried and kept at −20 °C for further analysis.

2.4. Color analysis

The color attributes of the isolated fucoidans were assessed using a microspectrophotometer (model 3/18, Sheen Instruments, UK). This instrument measures the light absorption and reflection properties of a sample to determine its color. For each fucoidan sample, three separate measurements were taken to ensure the reliability and accuracy of the results. The main color parameters measured are as follow: L* (brightness), a* (redness) and b* (yellowness).

2.5. Chemical analysis

The sulfate content was assessed using a modified BaCl₂-gelatin technique (Dodgson & Price, 1962). In summary, 2–3 mg of fucoidans was hydrolyzed in 3 mL of 0.5 M HCl at 120 °C for duration of 5 h. A 0.2 mL aliquot of the hydrolysate was then combined with 3.8 mL of 3 % trichloroacetic acid (TCA) and 1 mL of BaCl₂-gelatin reagent. Following a 20-min reaction interval, the absorbance was measured at 360 nm. K₂SO₄ used to draw the standard equation and the final computation was predicated on the SO₄²⁻ concentration. The amount of uronic acid was measured using a modified sulfamate/m-hydroxydiphenyl technique (Filisetti-Cozzi & Carpita, 1991). Fucoidan solutions were subjected to treatment with 2.4 mL of concentrated H₂SO₄ containing 0.075 M sodium tetraborate. Subsequently, 40 μL of 4 M sulfamic acid‑potassium sulfamate (pH 1.6) was added and the mixture was incubated at 100 °C for 20 min. Following cooling in an ice bath, 80 μL of 0.15 % (w/v) m-hydroxydiphenyl was introduced and the mixture was allowed to equilibrate at ambient temperature for 10 min prior to measuring the absorbance at 525 nm. Glucuronic acid served as the calibration reference.

2.6. Monosaccharide composition

Gas chromatography-mass spectrometry (GC–MS) analysis was used to determine the component monosaccharides of the extracted fucoidans. A 2 mg sample of each fucoidan was subjected to hydrolysis with 4 M trifluoroacetic acid (TFA) at 120 °C for 5 h. The hydrolysate was further reduced with sodium borodeuteride (NaBD₄) and acetylated using acetic anhydride, rendering it suitable for GC–MS analysis. The acetylated products were then injected into a GC–MS system (6890 N/MSD5973, Agilent Technologies, Santa Clara) fitted with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm). The identification of monosaccharides was accomplished by comparing the acquired data with a library of established standards.

2.7. Characterization of molecular properties

In order to determine the molecular properties of extracted polysaccharides, high-performance size exclusion chromatography with multi-angle laser light scattering and refractive index detection system (HPSEC-MALLS-RI) was used. Also, the column used for molecules separations was a TSK G5000PW (7.5 mm × 600 mm, Toso-Biosep, Montgomeryville, PA, USA). After the fucoidan sample was dissolved in distilled water, the analysis of molecular characteristics was performed by washing the polysaccharides with an aqueous solution of 0.15 M NaNO₃ and 0.02 % NaN₃ as the mobile phase at a flow rate of 0.4 mL/min. Finally, average molecular weight (M_w) and radius of gyration (R_g) were calculated using ASTRA 5.3 software (Wyatt Technology Corp).

The method put forward by You and Lim (You & Lim, 2000) was used to calculate the specific volume of gyration (SV_g) value.

$${\mathit{SV}}_g=4/\text{3π}{\left(R_g\times {10}^8\right)}^3/\left(M_w/\mathrm{N}\right)=2.522{R_g}^3/M_w$$

The units for M_w, R_g and SV_g are g/mol, nm and cm³/g, respectively, while N stands for Avogadro's constant (6.02 × 10²³/mol) in the given equation.

3. Determination of DPPH radical scavenging activity

Fucoidan samples were prepared at final concentrations of 0.25, 0.5 and 1.0 mg/mL by dissolving the appropriate amounts in distilled water. A 0.1 mM solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was freshly prepared in methanol. For each assay, 100 μL of the fucoidan solution was mixed with 100  μL of the DPPH solution in each well. The reaction mixtures were mixed briefly and then incubated in the dark at room temperature for 30 min to allow the reaction to reach equilibrium. After incubation, the absorbance of each mixture was measured at 517 nm using a microplate reader (Brand-Williams et al., 1995). A control sample was prepared by mixing 1  μL of distilled water with 1  μL of the DPPH solution. The DPPH radical scavenging activity was calculated using the following formula:

 % DPPH scavenging activity = [(A-control – A-sample) / A-control] × 100.

where A-control is the absorbance of the control sample and A-sample is the absorbance of the fucoidan-treated sample.

4. Determination of ABTS radical scavenging activity

Fucoidan samples were prepared in distilled water at final concentrations of 0.25, 0.5 and 1.0 mg/mL. The ABTS radical cation (ABTS•⁺) was generated by mixing a 7 mM ABTS stock solution with 2.45 mM potassium persulfate and allowing the mixture to react in the dark at room temperature for 16 h. The resulting ABTS•⁺ solution was then diluted with distilled water to obtain an absorbance of 0.70 at 734 nm. For the assay, 100 μL of each fucoidan solution was combined with 100 μL of the diluted ABTS•⁺ solution in each well. The mixtures were mixed briefly and incubated in the dark at room temperature for 30 min. Absorbance was measured at 734 nm using a microplate reader (Prior et al., 2005). The percentage inhibition of the ABTS radical was calculated using the following equation:

 % ABTS Scavenging activity = [(A-control – A-sample) / A-control] × 100.

where A-control is the absorbance of the diluted ABTS•⁺ solution without fucoidan and A-sample is the absorbance after the addition of the fucoidan sample.

5. Fe³⁺ reducing antioxidant power

The Fe³⁺ reducing antioxidant power of fucoidan samples was assessed at concentrations of 0.25, 0.5 and 1.0 mg/mL by the method of Oyaizu (1986) with modifications. Fucoidan solutions were prepared by dissolving the appropriate amounts of fucoidan in distilled water to achieve the desired final concentrations. A 1 mL aliquot of each fucoidan solution was mixed with 1 mL of phosphate buffer (0.2 M, pH 6.6) and 1 mL of a 1 % potassium ferricyanide (K₃Fe(CN)₆) solution. The reaction mixture was incubated at 50 °C for 20 min. After incubation, 1 mL of trichloroacetic acid (TCA) (10 %) was added to stop the reaction, and the mixture was centrifuged at 3000 rpm for 10 min. To each supernatant, 1 mL of distilled water and 0.2 mL of ferric chloride (FeCl₃) solution (0.1 %) were added. The absorbance of the resulting solution was measured at 700 nm using a UV–Vis spectrophotometer.

5.1. Proliferation of RAW264.7 cells

RAW264.7 murine macrophage cells were cultured in RPMI-1640 media supplemented with 10 % FBS under controlled conditions of 37 °C and 5 % CO₂. The cells were then transferred to 96-well microplates at a density of 1 × 10⁵ cells/mL, with each well containing 100 μL of the cell suspension. After a 24-h incubation period, polysaccharides at concentrations of 5, 25 and 50 μg/mL were added to the wells (100 μL each well) and the plates were incubated for a further 24 h. After the culture medium was removed, 120 μL of WST-1 reagent was added to each well and the plate was incubated for 4 h. Absorbance at 450 nm was quantified with an ELISA reader (Summat et al., 2024). The proportion of macrophage proliferation was determined using the following equation:

Macrophage proliferation (%) = (At/Ac) × 100

In the specified equation, At denotes the absorbance of the experimental group, while Ac indicates the absorbance of the control group.

5.2. Activation of RAW264.7 cells release nitric oxide

In an atmosphere of 5 % CO₂ and at 37 °C, RAW264.7 cells were cultured in RPMI-1640 media supplemented with 10 % FBS serum. After being transferred in a 96-well microplate at a density of 1 × 10⁵ cell/mL with a volume of 100 μL, the cells were then incubated for 24 h. Afterwards, fucoidans at concentrations of 5, 25 and 50 μg/mL or lipopolysaccharides from Escherichia coli at a concentration of 1 μg/mL were added to the microplates and allowed to incubate for an additional 24 h. A 100 μL aliquot of the culture medium was collected and mixed with an equivalent proportion of Griess reagent (Green et al., 1982). This mixture was thereafter maintained in a dark area for 10 min. The nitrite concentration was determined by referencing a NaNO₂ standard curve and the optical density at a wavelength of 540 nm was measured using an ELISA reader (EL-8000; BioTek Instruments, Winooski, VT, USA).

5.3. Analysis of CD86 expression

CD86 expression was analyzed using flow cytometry. Stimulated cells with either LPS or fucoidan were harvested, washed with phosphate-buffered saline (PBS) containing 10 %FBS. Cells were then incubated with fluorochrome-conjugated anti-CD86 antibodies (Thermo Fisher Scientific, USA) for 60 min at 4 °C in the dark. After staining, cells were washed twice with PBS containing 10 % FBS and resuspended in PBS for analysis. Data acquisition was performed using a CytoFLEX flow cytometer (Beckman Coulter, Inc., USA).

5.4. Statistical analysis

Data were analyzed using SPSS software (Version 16; SPSS Inc., Chicago, IL, USA). Results are expressed as mean ± standard deviation (SD) from at least three independent experiments. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed by Duncan's multiple-range post hoc test. Statistical significance was defined as p < 0.05. Graphs were generated using Sigma Plot (Version 15).

6. Results and discussion

6.1. Color analysis of fucoidans

Color is a critical factor that greatly affects how consumers perceive, choose and accept food products and significantly contributes to the visual appeal and often serves as a marker for attributes such as freshness, quality and taste. The visual impact of food largely depends on the color integrity of its ingredients and additives, which play a key role in enhancing the overall attractiveness of product and influencing consumer preferences. The images and color analysis of fucoidans extracted from S. assimile under different extraction methods are shown in Fig. 1. Fig. 1B illustrates varying intensities of light to dark brown colors for the extracted fucoidans. Typically, fucoidan exhibits colors ranging from yellow to light brown, with occasional occurrences of dark brown (Chaloshtori et al., 2023; Darem et al., 2025). These variations are largely determined by the presence and type of pigments such as carotenoids and chlorophyll entrapped within its structure during the extraction and isolation processes (Yip et al., 2014). The concentration and stability of these pigments, as well as the extraction conditions—such as temperature, pH and the presence of oxidizing agents—play a significant role in the final appearance of fucoidan (Saepudin et al., 2018; Yip et al., 2014).

Spectrometry analysis is a useful tool for characterizing the color properties of fucoidans using the Lab* color factors standardized by the International Commission on Illumination. It measures L* (lightness), a* (green-red) and b* (blue-yellow) to provide quantitative data on the visual attributes of samples. L* values range from 0 to 100, indicating overall lightness or darkness in which a value of 0 represents absolute black while a value of 100 represents absolute white. The color analysis (Fig. 2C, D and E) of the present fucoidans indicated larger L* and b* values for AS-A4 and AS-A8, suggesting a lighter and yellow color profile for polysaccharides obtained under conditions employing 0.025 M hydrochloric acid for longer hours (p < 0.05).

Fig. 2.

Monosaccharide composition profiles of fucoidans extracted using water (AS-D2) (A) and 0.025 M HCl (AS-A2, AS-A4, AS-A8) (B—D). Chromatograms were obtained from a GC–MS system equipped with an HP-5MS capillary column. HPSEC-MALLS-RI chromatograms of fucoidans extracted using water (AS-D2) (E) and 0.025 M HCl (AS-A2, AS-A4, AS-A8) (F—H). Separation was performed on a TSK G5000PW column at a flow rate of 0.4 mL/min.

6.2. Yield of fucoidans

The varying degrees of concentration, temperature and extraction duration are crucial in determining the yield and quality of the extracted polysaccharide. Table 1 presents the yield and chemical composition of fucoidans isolated under different extraction conditions. The extraction yield of fucoidan obtained by distilled water at 60 °C was 5.0 %, the amount of which increased up to 5.6 % in the acidic condition of AS-A2. Consequently, extending the duration of extraction to 4 and 8 h with 0.025 M hydrochloric acid at a constant temperature of 60 °C resulted in a notable increase in the yields of fucoidans to 6.2 and 6.9 %, respectively. While the extraction yields reached here are higher than those reported for Laminaria longipes (0.3 %, 0.1 N HCl, 1.5 h, 60 °C), Saccharina cichorioides (4.1 %, 0.1 N HCl, 1.5 h, 60 °C) and Sargassum fusiforme (3.9 %, water, 3 h, 50 °C), they are similar or lower to those obtained for fucoidans from Cystoseira indica (5.7 %, water, 2 h, 65 °C) and Fucus evanescens (9.0 %, 0.1 N HCl, 3 h, 60 °C) (Cumashi et al., 2007; Imbs et al., 2015; Liu et al., 2020; Usoltseva et al., 2019). Differences in fucoidan yield from seaweeds arise due to variations in the species, age, habitat of the seaweed and extraction conditions (e. g. solvent type, temperature and duration), which affect cell wall composition and fucoidan content (Jayawardena et al., 2022). The present results indicated that the utilization of dilute acidic condition notably improve the efficiency of polysaccharide recovery by plausibly affecting the disruption of cellular matrices and solubility and release of fucoidan.

Table 1.

Chemical composition and molecular weight of fucoidans extracted under different temperatures (2, 4 and 8 h) from S. assimile.^a

AS-A8	AS-A4	AS-A2	AS-D2
6.95 ± 0.27	6.21 ± 0.04	5.65 ± 0.35	5.00 ± 0.32		Yield (%)
17.46 ± 0.43	15.59 ± 0.46	16.55 ± 0.75	5.82 ± 0.50		Sulfate (%)
11.08 ± 0.32	9.46 ± 0.68	1.91 ± 0.83	1.96 ± 0.77		Uronic acid (%)
2.27 ± 0.01	2.42 ± 0.01	2.56 ± 0.02	2.97 ± 0.03	Rhamnose	Monosaccharide composition (%)
36.66 ± 0.05	35.25 ± 0.01	34.96 ± 0.570	32.92 ± 0.05	Fucose
5.21 ± 0.07	4.63 ± 0.02	4.80 ± 0.02	5.27 ± 0.06	Xylose
17.53 ± 0.01	18.45 ± 0.01	16.50 ± 0.06	17.62 ± 0.00	Mannose
11.58 ± 0.05	12.56 ± 0.01	15.27 ± 0.10	14.76 ± 0.19	Glucose
26.65 ± 0.14	26.65 ± 0.01	0.028 ± 25.87	26.3 ± 0.06	Galactose
1239.65 ± 6.86	1558.20 ± 5.37	1697.45 ± 9.12	1433.95 ± 18.74		Mw × 103 (g/mol)
48.60 ± 3.25	47.70 ± 0.56	60.10 ± 1.84	66.15 ± 1.13		Rg (nm)
0.23 ± 0.04	0.17 ± 0.00	0.32 ± 0.03	0.56 ± 0.03		SVg (cm3/g)

^aExtraction conditions included aqueous (AS-D2: 60 °C, 2 h) and acidic (AS-A2, AS-A4, AS-A8: 0.025 M HCl, 2–8 h, 60 °C).

6.3. Chemical composition of fucoidans

The chemical compositions of fucoidans are shown in Table 1. As the present results suggest, the fucoidans isolated from S. assimile possess an anionic character owed to the presence of sulfates (5.8 to 17.4 %) and uronic acids (1.9 to 11.0 %). Fucoidans isolated using distilled water contained the lowest amounts of sulfates and uronic acids in comparison with acidic conditions in which they both considerably increased with rising extraction times. The sulfate content in fucoidans can range from approximately 6.0 to 38.3 % by weight (Bilan et al., 2004; Daub et al., 2020). Some less sulfated fucoidans may fall slightly below this range for example the one from Sargassum sp. (4.7 %), while some highly sulfated ones can significantly exceed this and reach as high as 46.5 % in Fucus evanescens (Bilan et al., 2004; Lutfia et al., 2020). In the current results, enhanced sulfate content in fucoidans extracted using dilute acid, compared to water, may result from the selective solubilization of sulfated fractions (Dobrinčić et al., 2021). Mechanistically, the acidic environment can disrupt hydrogen bonds and ionic interactions within the seaweed cell wall matrix, preferentially releasing highly sulfated fucoidan molecules (Dobrinčić et al., 2021). Additionally, acid hydrolysis can partially cleave non-sulfated polysaccharide impurities, enriching the relative proportion of sulfated fucoidan in the extract. This selective extraction increases the apparent sulfate content despite potential desulfation effects which could be more common in stronger acidic conditions (Chaloshtori et al., 2023).

Fucoidan chains were composed of a diverse array of sugars, with fucose (32.9 to 36.6 %) and galactose (25.8 to 26.6 %) being the most prevalent monosaccharides and mannose, glucose, xylose and rhamnose were present in lesser amounts (Fig. 2A-D and Table 1). The amounts of fucose monosaccharides in fucoidan samples increased from distilled water to acidic conditions and with prolonged extraction durations (Table 1). Conversely, glucose levels were the highest in AS-D2 and AS-A2 fucoidans, while significantly diminishing with prolonged extraction durations. In line with the present results, type of solvent and extraction time are found to be strongly affecting the fucose contents in fucoidans isolated from brown seaweeds (Dobrinčić et al., 2021). Considerably larger levels of fucose were contained in fucoidans extracted using 0.1 M HCl rather than water for Fucus virsoides (27.9 to 41.5 %) and Cystoseira barbata (11.7 to 20.9 %) (Dobrinčić et al., 2021). However, the extraction time exhibited inconsistent findings; although its extension led to increased fucose in C. barbata, when investigated in F. virsoides it resulted in reduced fucose amount (Dobrinčić et al., 2021). In a recent work, we showed that raising HCl concentration from 0.025 M to 0.2 M at room temperature enhances the amount of fucose (39.9 to 45.8 %) involved in fucoidan composition of C. indica (Chaloshtori et al., 2023). Nevertheless, the effect of temperature was strongly related on acid concentration; whereas, in more diluted HCl concentrations, the amount of fucose increased with enhanced temperature, in more concentrated HCl conditions, the fucose content dramatically decreased at higher temperatures (Chaloshtori et al., 2023). The reduction in fucose content under elevated temperature and acid concentration is due to acid-catalyzed hydrolysis of glycosidic bonds, leading to fucoidan depolymerization and the release of smaller oligosaccharides or monosaccharides (Baba et al., 2018).

6.4. Molecular properties of fucoidans

The molecular weight distribution of polysaccharides extracted in different conditions was analyzed using an HPSEC-MALLS-RI system, with the outcomes summarized in Fig. 2E-H and Table 1. The results revealed that all extracted polysaccharides formed a heterogeneous mixture, comprising three distinct polymer fractions with varying molecular weights. The first fraction consisted of high molecular weight polysaccharides, while the second and third fractions had medium to low molecular weight polymers. The slight shift in elution profiles of isolated fucoidans suggests that the extraction solvent and time played a significant role in determining the molecular weight distribution of the polysaccharides. The analysis indicated that the fucoidan extracted using water had a high M_w of 1433.9 × 10³ g/mol. When acidic condition was used, considerably larger polysaccharide chains with higher M_w of 1697.4 × 10³ g/mol were isolated from the cell wall matrix. Increasing the duration of exposure of fucoidans to an acidic environment at 60 °C appears to promote the disintegration of polymer chains, which gradually lowered their M_w to 1239.6 × 10³ g/mol. Previously, it was indicated that increasing acid concentration and extraction temperature in fucoidan extraction significantly reduce the molecular weight of fucoidan, with higher temperatures (80 °C) and acid concentrations (0.2 M HCl) causing notable degradation. In contrast, lower acid concentrations (0.025–0.05 M) and room temperature preserved larger molecular weights (up to 1990.0 × 10³ g/mol) (Chaloshtori et al., 2023). In a different study, it was found that extended extraction time in acid significantly reduces the molecular weight of fucoidan, with rapid hydrolysis observed initially (30 % reduction in 6 min) and continued degradation over 180 min (86–88 % reduction) (Lorbeer et al., 2015). The decline followed an exponential trend, indicating diminishing returns in structural integrity with prolonged extraction. More research into the structure of fucoidans revealed that their R_g varied, ranging from 47.7 to 66.15 nm. The R_g of a molecule quantifies the dispersion of the molecule relative to its center of mass and is often used to characterize the dimensions and morphology of macromolecules, particularly regarding biopolymers (Tabarsa et al., 2019). These results showed that the fucoidan molecules were of different sizes and employing the acidic condition reduced the size of the molecules in an adverse correlation with the extraction time. The SV_g of polysaccharides reflects the spatial volume occupied by the polymer in solution, normalized to its molecular weight and provides insight into the spatial distribution and density of the polymer in solution (Tabarsa et al., 2019). The SV_g values of the present fucoidans varied between 0.17 and 0.56 cm³/g revealing that polysaccharide molecules obtained using water had more expanded conformation compared to those extracted under acidic conditions. Water extraction likely preserves or promotes a more hydrated and expanded conformation of fucoidans by acting as a proper solvent that facilitates polymer chain unfolding, whereas acidic extraction may cause partial hydrolysis or glycosidic bond breakage, reducing molecular weight, altering ionic interactions and compacting the polysaccharide structure into a more collapsed or dense conformation.

6.5. Antioxidant properties of fucoidans

Antioxidant assays are essential for evaluating the ability of compounds to neutralize free radicals, which can cause oxidative stress and damage in biological systems. Three commonly used assays are DPPH radical scavenging activity, ABTS radical scavenging activity and reducing power (Munteanu & Apetrei, 2021). In the DPPH assay, antioxidants donate electrons or hydrogen atoms to the DPPH radical, reducing its purple color to yellow, with absorbance measured at 517 nm (Munteanu & Apetrei, 2021). In the present study, antioxidant activities of fucoidans were evaluated over the concentrations of 0.25, 0.5 and 1.0 mg/mL and the results are presented in Fig. 3. Fucoidans showed significantly-varied scavenging effects on DPPH free radicals ranging from 30.5 to 48.4 % (Fig. 3A). The activity of all samples was dose-dependent, increasing with concentration from 0.25 to 1.0 mg/mL (p < 0.05). At 0.25 mg/mL, scavenging activities ranged from 30.5 to 36.8 %, while at 1.0 mg/mL, activities increased but remained below 50 %, with AS-D2 exhibiting the highest activity (48.4 %, EC₅₀ 1.23 mg/mL, p < 0.05). Samples extracted with dilute acid showed slightly lower activities, particularly with longer extraction times, as seen with AS-A4 and AS-A8, which demonstrated reduced scavenging potential across all concentrations compared to AS-D2. The standard antioxidant, ascorbic acid (0.05 mg/mL), exhibited significantly higher activity (63.018 %, EC₅₀ 0.037 mg/mL), highlighting the relatively moderate efficacy of the fucoidans. The ABTS assay generates the ABTS radical cation, which is reduced by antioxidants, leading to a color change from blue-green to colorless, measured at 734 nm (Munteanu & Apetrei, 2021). The ABTS radical scavenging activity of fucoidans was evaluated and the results are presented in Fig. 3B. All samples demonstrated a dose-dependent increase in scavenging activity. At 0.25 mg/mL, AS-D2 exhibited the highest activity (58.9 %, EC₅₀ 0.59 mg/mL), while samples extracted with acid showed slightly lower activities, decreasing with longer extraction durations. At 0.5 mg/mL, scavenging activities were more consistent across all samples, ranging from 78.8 to 81.6 %. At 1.0 mg/mL, the scavenging activity reached approximately 89 % for all samples. The standard antioxidant, ascorbic acid, displayed the highest activity (90.8 %, EC₅₀ 0.029 mg/mL) at 0.05 mg/mL, surpassing the tested fucoidans even at higher concentrations. The reducing power assay involves the reduction of ferric ions (Fe³⁺) to ferrous ions (Fe²⁺), producing a colored complex quantified at 700 nm. The results of ferric reducing power of fucoidans are presented in Fig. 3C. All samples demonstrated dose-dependent ferric reducing activity in concentrations of 0.25 to 1.0 mg/mL which ranged between 0.16 and 0.26 (Abs). The results indicated nearly no significant differences in the capacities of different fucoidans in reducing Fe³⁺ to Fe²⁺ ions (p > 0.05). The standard antioxidant, ascorbic acid, showed significantly greater reducing power (Abs 0.96) at 0.05 mg/mL, emphasizing its superior efficacy.

Fig. 3.

DPPH (A), ABTS (B) radical scavenging activities, ferric reducing power (C), RAW264.7 murine macrophage proliferation (D) and nitric oxide production (E) of fucoidans extracted using water (AS-D2) and 0.025 M HCl (AS-A2, AS-A4, AS-A8). RAW264.7 cells (1 × 10⁵ cells/mL) were cultured in RPMI-1640 media supplemented with 10 % FBS under controlled conditions of 37 °C and 5 % CO₂ and incubated with 100 μL of samples. Superscript small letters indicate significant differences between fucoidans (p < 0.05). Ascorbic acid (AA) and lipopolysaccharide (LPS) were used as positive controls.

6.6. Immunostimulatory effects of fucoidans

Different concentrations of fucoidans (5, 25 and 50 μg/mL) were added to RAW 264.7 macrophages and incubated at 37 °C under 5 % CO₂ for 24 h. Cell viability, assessed using the WST-1 assay as an indicator of cellular metabolic activity, confirmed that none of the fucoidans isolated under different extraction conditions exerted cytotoxic effects on macrophages (p > 0.05) compared with untreated controls (Fig. 3D). The consistently high viability (> 96 %) across all treatments demonstrates that the concentrations used in subsequent nitric oxide assays were physiologically safe and that the observed immunostimulatory responses reflected genuine bioactivity rather than cytotoxic stress (Fig. 3D). Nitric oxide (NO), a gaseous signaling molecule generated by macrophages through inducible nitric oxide synthase (iNOS), functions as a critical effector molecule in innate immunity, contributing to the cytotoxic eradication of microbial pathogens and neoplastic cells (Tripathi et al., 2007). The effect of different fucoidan extraction methods on NO production in RAW264.7 cells was evaluated by exposing cells to various concentrations (5, 25 and 50 μg/mL) of fucoidans extracted under distinct conditions. As shown in Fig. 3E, NO production increased in a dose-dependent manner for all extraction methods. At the highest concentration (50 μg/mL), all fucoidan preparations induced the highest levels of NO production (p < 0.05), exceeding that of LPS used as positive control. Among the different extraction methods, fucoidans extracted with hydrochloric acid for 8 h (AS-A8) demonstrated the highest nitric oxide production, particularly at 50 μg/mL, followed by those extracted with 4 h of hydrochloric acid (AS-A4) (p < 0.05). Fucoidans extracted with water for 2 h (AS-D2) and hydrochloric acid for 2 h (AS-A2) also produced increased nitric oxide levels, although to a lesser extent compared to the longer acid extraction times. In line with the present results, our previous studies also demonstrated significant release of NO from macrophage cells induced by unrefined fucoidans extracted from C. indica, Padina australis, Nizamuddinia zarnardinii (Chaloshtori et al., 2023; Darem et al., 2025; Mousavi et al., 2024). It has been shown that fucoidans obtained under mild acid extraction could exhibit markedly higher NO-releasing activity compared with those extracted with water. For instance, in C. indica, 0.05–0.1 M HCl at 60–80 °C enhanced fucoidan yield while optimizing sulfation and reducing molecular weight—features linked to stronger RAW264.7 macrophage activation through NF-κB and MAPKs pathways (Chaloshtori et al., 2023). Similarly, acid-extracted fucoidan from N. zanardinii (0.01 N HCl, 20 min) significantly increased NO production compared with native water-extracted samples but yet lower than those isolated using enzymes (Darem et al., 2025). Collectively, these findings demonstrate that controlled acid extraction not only tailors molecular size of fucoidan but also preserves key sulfate groups, thereby producing structurally optimized fractions with superior nitric oxide–mediated immunostimulatory efficacy.

6.7. Chemical and monosaccharide compositions of fucoidan fractions

Given the high extraction yield, favorable chemical composition (sulfate and fucose contents) and superior antioxidant and macrophage stimulating capacities, fucoidan isolated using 0.025 M hydrochloric acid for 8 h (AS-A8) was chosen for further fractionation based on molecular weight. Table 1 presents the chemical compositions of fucoidan fractions obtained through a cross-flow ultrafiltration system with 2, 10 and 30 kDa M_w cut-offs. The findings indicated that these fucoidan fractions contain varying levels of sulfates (14.6 to 16.2 %) and uronic acids (5.0 to 17.4 %). The FT-IR spectra of the samples (Suppl. 1) indicated intense absorption peaks across 400–4000 cm⁻¹ for all sample. The spectral features observed particularly in the 800–1200 cm⁻¹ region and near 3400 cm⁻¹ correspond well with the typical vibrational bands associated with polysaccharide structures and fucoidans (Borazjani et al., 2018). The broad, intense band near 3400 cm⁻¹ was assigned to O—H stretching vibrations. As expected for sulfated polysaccharides, two prominent absorptions were also detected: one around 850 cm⁻¹, reflecting the S—O stretching of sulfate groups, and another near 1250 cm⁻¹, associated with axial C–O–S bending vibrations (Alboofetileh et al., 2019).

The monosaccharide composition of fucoidan fractions obtained through ultrafiltration using 2, 10 and 30 kDa M_w cut-offs revealed distinct sugar distributions (Fig. 4A-C and Table 2). Fucose was the dominant sugar, with the highest content in the AS-A8–30 < kDa fraction (39.93 %), decreasing in lower M_w fractions, indicating preferential retention of fucose-rich polysaccharides in higher M_w fractions. Rhamnose and xylose were present in minor amounts, with rhamnose slightly increasing in smaller M_w fractions. Mannose levels remained stable, while glucose was significantly enriched in lower M_w fractions, suggesting co-extraction of laminarin as glucose-rich polysaccharides. Galactose content varied, being highest in AS-A8–30 < kDa and lower in intermediate fractions. These trends align with previous research, suggesting that M_w influences the structural and functional properties fucoidan.

Fig. 4.

Monosaccharide composition profiles of fractionated fucoidans including AS-A8–2-10 kDa (A), AS-A8–10-30 kDa (B) and AS-A8–30 < kDa (C). Chromatograms were obtained from a GC–MS system equipped with an HP-5MS capillary column. Molecular properties of fractionated fucoidans including AS-A8–2-10 kDa (D), AS-A8–10-30 kDa (E) and AS-A8–30 < kDa (F) analyzed by HPSEC-MALLS-RI.

Table 2.

Chemical composition and molecular weight of purified fractions obtained from fucoidan extracted for 8 h using 0.025 M hydrochloric acid (AS-A8)^a.

AS-A8-30 < kDa	AS-A8–10-30 kDa	AS-A8–2-10 kDa
0.61 ± 15.50	0.79 ± 16.24	0.53 ± 14.69		Sulfate (%)
15.63 ± 0.05	5.06 ± 0.68	17.36 ± 0.10		Uronic acids (%)
2.41 ± 0.01	2.02 ± 0.05	3.13 ± 0.10	Rhamnose	Monosaccharide
composition (%)
0.01 ± 39.93	0.02 ± 23.82	0.15 ± 20.86	Fucose
0.06 ± 4.84	0.00 ± 4.82	0.01 ± 3.64	Xylose
0.06 ± 16.97	0.06 ± 16.67	0.23 ± 16.63	Mannose
0.03 ± 8.70	0.05 ± 35.23	0.10 ± 36.20	Glucose
0.11 ± 27.11	0.03 ± 17.40	0.08 ± 19.49	Galactose
715.05 ± 69.79	23.50 ± 0.70	6.60 ± 0.98		Mw × 103 (g/mol)
33.85 ± 2.47	75.50 ± 0.56	79.95 ± 0.21		Rg (nm)
0.13 ± 0.01	46.19 ± 0.35	197.38 ± 28.05		SVg (cm3/g)

^aExtraction conditions was acidic (AS-A8: 0.025 M HCl, 8 h, 60 °C).

6.8. Molecular properties of fucoidan fractions

The molecular properties of fucoidan fractions obtained through ultrafiltration using 2, 10 and 30 kDa molecular weight cut-offs exhibited significant differences in M_w, R_g and SV_g (Fig. 4D-F and Table 2). The highest M_w was observed in the AS-A8–30 < kDa fraction (715.05 × 10³ g/mol), while lower M_w values were recorded for the AS-A8–10-30 kDa (23.50 × 10³ g/mol) and AS-A8–2-10 kDa (6.60 × 10³ g/mol) fractions. This trend suggests that high-M_w fucoidan components were retained in the larger cut-off fraction, while smaller M_w fractions contained more fragmented polysaccharides. The R_g, which reflects molecular conformation and flexibility, exhibited an increasing trend with decreasing M_w. The AS-A8–30 < kDa fraction had the smallest R_g (33.85 nm), whereas the AS-A8–10-30 kDa and AS-A8–2-10 kDa fractions displayed significantly larger values (75.50 nm and 79.95 nm, respectively). The SV_g also varied substantially among the fractions, with the lowest value in AS-A8–30 < kDa (0.13 cm³/g), followed by a notable increase in AS-A8–10-30 kDa (46.19 cm³/g) and a dramatic rise in AS-A8–2-10 kDa (197.38 cm³/g). This suggests that the lower M_w fractions adopted more expanded conformations, possibly due to a higher degree of branching.

6.9. Antioxidant properties of fucoidan fractions

The DPPH radical scavenging activity of fucoidan fractions obtained through ultrafiltration using 2, 10 and 30 kDa M_w cut-offs demonstrated concentration-dependent antioxidant potential (Fig. 5A). The scavenging activity increased with increasing sample concentration (0.25, 0.5 and 1.0 mg/mL) in all fractions (p < 0.05). Among the fucoidan fractions, AS-A8–2-10 kDa exhibited the highest radical scavenging activity (61.19 %; EC₅₀ 0.054 mg/mL), followed by AS-A8–10-30 kDa (55.88 %) and AS-A8–30 kDa (46.22 %), suggesting that lower M_w fractions possess enhanced antioxidant properties (p < 0.05). The EC₅₀ of ascorbic acid for scavenging DPPH radicals is 0.037 mg/mL. Similarly, ABTS radical scavenging activity (Fig. 5B) followed a comparable trend, with AS-A8–2-10 kDa exhibiting the highest scavenging ability (97.99 %, EC₅₀ 0.154 mg/mL), particularly at 1.0 mg/mL. The AS-A8–10-30 kDa (69.30 %) and AS-A8–30 kDa (58.59 %) fractions showed lower scavenging efficiencies, reinforcing the correlation between molecular weight and antioxidant capacity (p < 0.05). The EC₅₀ of ascorbic acid for scavenging ABTS radicals is 0.029 mg/mL. The reducing power assay (Fig. 5C) also demonstrated that AS-A8–2-10 kDa had the highest reducing power (Abs 0.26), followed by AS-A8–10-30 kDa (Abs 0.17) and AS-A8–30 kDa (Abs 0.17) (p < 0.05).

Fig. 5.

DPPH (A), ABTS (B) radical scavenging activities, ferric reducing power (C), RAW264.7 murine macrophage proliferation (D) and nitric oxide production (E) of fractionated fucoidans (AS-A8–2-10 kDa, AS-A8–10-30 kDa, AS-A8–30 < kDa). RAW264.7 cells (1 × 10⁵ cells/mL) were cultured in RPMI-1640 media supplemented with 10 % FBS under controlled conditions of 37 °C and 5 % CO₂ and incubated with 100 μL of samples. Superscript small letters indicate significant differences between fucoidans (p < 0.05). Ascorbic acid (AA) and lipopolysaccharide (LPS) were used as positive controls.

6.10. Immunostimulatory properties of fucoidan fractions

The proliferation effects of fucoidan fractions on RAW264.7 macrophage cells were evaluated at different concentrations (5, 25, and 50 μg/mL). The results indicated that fucoidan fractions did not exhibit significant cytotoxic effects on macrophages at the tested concentrations, as the cell viability remained above 96 % in all conditions. The effects of fucoidan fractions on NO production in RAW264.7 macrophages were assessed at the same concentrations. The results demonstrated that fucoidan fractions influenced NO production in a dose-dependent manner (p < 0.05). At 50 μg/mL, all fractions showed a significant increase, with AS-A8–10-30 kDa producing the highest NO levels (26.54 μM), followed by AS-A8–2-10 kDa (25.14 μM) and AS-A8–30 kDa (22.77 μM). The LPS-stimulated control exhibited high NO production (23.59 μM), which was comparable to the levels observed in the highest concentration of fucoidan fractions. These findings suggest that fucoidan fractions can modulate NO production in macrophages, with fractions of 10–30 kDa generally exhibiting greater activity.

The pathophysiology of inflammatory processes has been linked to complement receptor CD86, a protein that is often expressed on antigen-presenting cells including macrophages (Chen et al., 2022). Its importance in controlling T lymphocyte activation, proliferation and effector function is highlighted by its function as a costimulatory molecule for T cell activation. The present findings suggested that AS-A8–10-30 kDa fucoidan fraction would have induced the production of inflammatory mediators by RAW264.7 cells. This activation most likely happens via the production of the NF-κB and MAPK signaling pathways as well as the cell surface receptor of CD86 (Fig. 6).

Fig. 6.

CD86 expression levels in untreated RAW264.7 cells (A), cells incubated with LPS (B), cells incubated with AS-A8–10-30 kDa (C) and their quantitative analysis (D). Cells were incubated with fluorochrome-conjugated anti-CD86 antibodies for 60 min at 4 °C in the dark. Superscript small letters indicate significant differences between treatments (p < 0.05).

6.11. Structure-activity relationship

Monosaccharide composition, amount of sulfate esters, content of uronic acids, molecular weight and conformation are the main structural attributes of fucoidans proposed as being the determinant factor of their biological activities (Supanto et al., 2019; Zhang et al., 2014). Herein, the presence of certain amounts of sulfates esterified on polysaccharide chains and an optimum molecular weight have been deemed necessary for exerting the basic biological functions (Fukahori et al., 2008; Saepudin et al., 2018). Fucoidans exhibit strong antioxidant properties in which sulfate groups play a crucial role by enhancing their ability to donate electrons and stabilize free radicals. For instance, sulfonation of fucoidan from Sargassum filipendula increased its sulfate content by 3 %, which resulted in higher antioxidant activity compared to crude fucoidan (Supanto et al., 2019). Similarly, when the sulfate content of fucoidan from Sargassum siliquosum increased from 18.7 to 32.1 %, the antioxidant activity, as indicated by a decrease in EC50 values for DPPH scavenging, improved significantly (Chen et al., 2025). Molecular weight also influences antioxidant activity, with lower molecular weight fucoidans often exhibiting better radical-scavenging ability and metal ion chelation due to improved solubility and bioavailability. For instance, fucoidans degraded by gamma ray irradiation showed increased ferric-reducing antioxidant power from 0.137 to 1.754 mM as their molecular weight decreased from 217.0 to 7.0 kDa (Lim et al., 2014). Additionally, fucoidans subjected to sonochemical treatment exhibited a significant decrease in molecular weight (from 815 kDa to 318 kDa) and a corresponding increase in antioxidant activity (88.9 % compared to 65.3 % in the control process) (Bagale et al., 2023). In contrast, fucoidans with higher molecular weights were also found to have a positive correlation with reducing power, which are indicators of antioxidant activity (Qu et al., 2014). It is worth noting that the relationship between molecular weight and antioxidant activity is not simply linear; nonetheless, in the current study, lower-molecular-weight fucoidans exhibited stronger antioxidant activities (Supple. 2). One study highlighted that fucoidans with molecular weights of 3.8 kDa, 1.0 kDa and > 8.3 kDa exhibited better antioxidant activities in different assays, suggesting that both very low and relatively high molecular weight fucoidans can be effective antioxidants (Hou et al., 2012).

The immunostimulatory effects of fucoidan are significantly influenced by its sulfate content. Several studies have demonstrated that the presence and degree of sulfation in fucoidans are critical for their ability to modulate immune responses. Fucoidan promotes the activation and proliferation of lymphoid cells, including NK cells, T cells and dendritic cells and enhances the expression of activation markers such as CD40, CD80, and CD86 which is linked to the presence of sulfate groups (Jin et al., 2014; Lebedynskaya et al., 2015). Fucoidan enhances the production of IL-12 and IFN-γ in response to lactic acid bacteria and this activity is abolished when fucoidan is desulfated, further underscoring the role of sulfate groups in its immunostimulatory effects (Kawashima et al., 2012). Both low and high molecular weight fucoidans exhibit significant immunostimulatory effects, but their mechanisms and impacts differ. High molecular weight fucoidans generally increase the production of a broader range of cytokines, enhancing immune responses, whereas low molecular weight fucoidans effects are more targeted, often reducing pro-inflammatory cytokines through specific signaling pathways (Chen et al., 2022; Jang et al., 2014; Liu et al., 2025). While our results demonstrate a clear dependence of NO production on fucoidan molecular weight and sulfation, the signaling receptors mediating this immunostimulatory response remain to be confirmed (Supple. 2). Several studies implicate pattern-recognition receptors in fucoidan sensing: TLR4 (and in some cases TLR2) has been reported to mediate polysaccharide-driven macrophage activation and downstream NF-κB/MAPK signaling, consistent with elevated iNOS and NO release (Chaloshtori et al., 2023; Hsu et al., 2017). In parallel, class A and B scavenger receptors (e.g., SR-A/CD204, CD36) have been identified as direct fucoidan ligands and shown to contribute to macrophage activation in several model systems, providing an alternative or complementary pathway for NO induction (Cui et al., 2022) Based on these reports, we hypothesize that the medium-range molecular weight (10–30 kDa) and preserved sulfation of AS-A8–10-30 kDa enhance binding to macrophage surface receptors (TLR4 and/or scavenger receptors), thereby triggering MyD88-dependent MAPK and NF-κB pathways that upregulate iNOS expression and NO release (Zayed et al., 2023).

7. Conclusions

This study elucidated the structure–function relationship of fucoidans extracted from Sargassum assimile under different chemical and physical conditions. Comparative extraction revealed that dilute acid treatments enhanced yield and altered the molecular and compositional profiles of fucoidans, while prolonged exposure decreased molecular weight and modified polymer conformation. Fractionation by ultrafiltration further demonstrated that molecular weight plays a decisive role in bioactivity: lower-molecular-weight fucoidans exhibited stronger antioxidant activities, whereas medium-weight fractions induced higher nitric oxide production in macrophages, suggesting distinct functional pathways. Overall, sulfate content, molecular weight and conformation collectively influenced the antioxidant and immunostimulatory functions of fucoidans, with no single factor acting independently. These findings contribute to a deeper understanding of the structure–activity correlations governing marine polysaccharides and provide a basis for optimizing extraction strategies to tailor fucoidan functionality for nutraceutical and pharmaceutical applications. Additionally, this research promotes the sustainable utilization of marine biomass by converting S. assimile, into bioactive compounds with potential health and industrial applications. Beyond laboratory optimization, the mild acid extraction and cross-flow ultrafiltration techniques described here are readily adaptable to industrial-scale operations, offering cost-efficiency and process reproducibility. The resulting fucoidan fractions demonstrate structural stability under ambient storage conditions, ensuring functional integrity during handling and formulation. These features are crucial for the development of nutraceutical and pharmaceutical products where batch consistency, bioactivity retention, and shelf-life are essential for market translation. By valorizing S. assimile into bioactive fucoidan fractions using mild and eco-friendly processes, this study supports circular resource utilization, fostering innovation and sustainability in the blue bioeconomy through the development of high-value marine-derived functional ingredients.”

CRediT authorship contribution statement

Kimia Sahragard: Writing – original draft, Methodology, Investigation. Mehdi Tabarsa: Writing – review & editing, Writing – original draft, Supervision, Investigation. Hassan Ahmadi Gavlighi: Writing – review & editing, Methodology. Amir Pouya Ghandehari Yazdi: Writing – review & editing, Methodology.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary 1. FT-IR spectra of AS-A8-2-10 kDa (A), AS-A8-10-30 kDa (B) and AS-A8-30 < kDa (C) fucoidans. Supplementary 2. Bubble plot of the effect of sulfate and molecular weight of fucoidans on DPPH (A), ABTS (B) and NO (C) activities.

Data availability

Data will be made available on request.