Introduction

Brown seaweeds represent an abundant and underexplored marine resource along the Indian coastline, offering a rich repository of bioactive compounds with considerable health and commercial potential [1,2]. Among these, fucoidan, a sulfated polysaccharide primarily found in the cell walls of brown algae such as Fucus, Sargassum, Laminaria, and Turbinaria, has garnered significant scientific interest due to its broad spectrum of biological activities [3,4]. Structurally, fucoidans are complex heteropolysaccharides composed predominantly of α-L-fucopyranose units, often linked with other sugar monomers such as glucose, galactose, mannose, uronic acids, and sulfate groups features that are closely associated with their bioactivity [5]. Numerous studies have highlighted fucoidan’s antioxidant, antidiabetic, anticancer, anti-inflammatory, antimicrobial, and anticoagulant properties, making it a promising candidate for applications in pharmaceuticals, nutraceuticals, and functional foods [6,7]. These biological activities vary depending on the seaweed species, harvesting conditions, geographic location, and extraction methods [8]. These biological activities vary depending on the seaweed species, harvesting conditions, geographic location, and extraction methods. However, regional studies focusing on the bioactivity of fucoidan from Indian brown seaweeds remain limited, particularly those examining species from the biodiverse Pudumadam coast in the Gulf of Mannar region, Tamil Nadu.

Chronic diseases such as diabetes mellitus and cancer are on the rise globally, with oxidative stress playing a central role in their pathophysiology [9]. Type 2 diabetes, in particular, is associated with elevated postprandial glucose levels due to the action of digestive enzymes such as α-amylase and α-D-glucosidase [10,11]. Synthetic inhibitors of these enzymes are widely used but often have limitations, including undesirable side effects and limited efficacy. Similarly, traditional cancer therapies can result in significant systemic toxicity and poor patient outcomes. These challenges underscore the need for safe, natural bioactive agents capable of modulating oxidative stress and inhibiting disease-related pathways [10,12].

In this context, fucoidans derived from marine brown algae offer a sustainable and effective therapeutic alternative. This study aims to characterize fucoidan extracted from six brown seaweed species (Sargassum wightii, Sargassum tenerrimum, Sargassum cinereum, Turbinaria conoides, Padina boergesenii, and Padina tetrastromatica) collected from the Pudumadam coast, Gulf of mannar region, Tamil Nadu, India. The biological potential of the extracted fucoidans was assessed using multiple in vitro assays, including antioxidant (DPPH, FRAP and HRSA), antidiabetic (α-amylase and α-D-glucosidase inhibition), and anticancer (cytotoxicity against MCF-7 cells) evaluations. The findings contribute to the growing body of evidence supporting marine algal polysaccharides as multifunctional bioactive agents and emphasize the relevance of India’s coastal biodiversity in developing natural health-promoting candidates for chronic disease management.

Furthermore, Principal Component Analysis (PCA) was applied to examine the relationships among the different biological activities and to identify major patterns of variation among the six seaweed species. This multivariate method helped group the species according to their antioxidant, antidiabetic and other biological potentials providing an integrated understanding of their functional diversity. The analysis also offered clearer insights into species-specific bioactivity patterns and supported a better interpretation of the overall experimental results.

Materials and methods

Determination of antioxidant capacity

DPPH scavenging activity.

The assay was performed in triplicate (n = 3) following the method of [17,18], with minor modifications. A 1.5 mL aliquot of methanolic DPPH solution (0.1 mM; Extrapure, 95% grade; SRL – Sisco Research Laboratories Pvt. Ltd., Product Code 29128, SDS Version 1.4; Taloja, Maharashtra, India) was mixed with 75 µL of fucoidan extract at various concentrations (2.0, 1.0, 0.5, 0.25, and 0.125 mg/mL). The mixtures were incubated at room temperature in the dark for 30 min. A standard calibration curve was prepared using L-ascorbic acid (purity 99%, SQ grade; Thermo Fisher Scientific Pvt. Ltd., Product No. 18455, SDS Version 3.0; Mumbai, Maharashtra, India) at concentrations ranging from 5 to 200 µg/mL. A mixture containing methanol and DPPH solution (without sample) served as the control, while methanol as the blank. Absorbance was measured at 517 nm using a UV–Visible spectrophotometer (Model DU 730, Beckman Coulter Inc., Brea, California, USA). Antioxidant activity was expressed as IC₅₀, defined as the concentration of extract required to inhibit 50% of the initial DPPH radical concentration. IC₅₀ values were calculated from the regression equation obtained by plotting the percentage of inhibition against sample concentration.

where A₀ was the absorbance of the control (DPPH solution without sample) and Aₛ was the absorbance of the sample. The IC₅₀ value was determined from the regression equation plotted between percentage inhibition and sample concentration.

ABTS scavenging activity.

This assay evaluates the ability of antioxidants to neutralize the generated ABTS radicals. Equal volumes of 7 mM ABTS (extra pure 98% HPLC grade, SRL-Sisco Research Laboratories Pvt. Ltd., P Code-28042, SDS version 4.0, Taloja, Maharashtra, India) and 2.45 mM Potassium persulphate (extra pure 98% AR grade, SRL-Sisco Research Laboratories Pvt. Ltd., P Code-48319, SDS version 4.0, Taloja, Maharashtra, India) were mixed to prepare the stock solution. The mixture was incubated in the dark for 12 h at room temperature (RT) to generate ABTS⁺ free radicals. Each time the working solution was freshly prepared by diluting the stock with 50% of methanol (purity-99.5% GC grade, Thermo Fisher Scientific- Pvt. Ltd, Product No.-32405, SDS version-2.0, Mumbai, Maharashtra, India) to obtain an absorbance of 0.70 ± 0.02 at 745nm and 30°C. Subsequently, 1200 µL of ABTS working solution was mixed with 120 µL of fucoidan extract at different concentrations (2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml), followed by incubation for 6 min at 30°C at 745 nm. The absorbance was recorded at 745 nm against methanol as the blank, using quercetin as the standard. The results were expressed as mean ± standard deviation (SD) from three independent replicates (n = 3) and the IC₅₀ values were calculated as described earlier [19].

HRSA scavenging activity.

The activity was tested as per [20]. A 500 µl extract of different concentration (2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml) was taken and 500 µl of 1.5mM Ferrous sulfate (purity-98% SQ grade, Thermo Fisher Scientific Pvt. Ltd, Product No.-23755, SDS version-2.0, Mumbai, Maharashtra, India), 350 µl of 6 mM H₂O₂ (50%, EMPLURA®, Merck Life Sciences Pvt. Ltd, Material code 1.93408.0521, Mumbai, Maharashtra, India) and 150 µl of Sodium salicylate (purity-99.5% SQ grade, Thermo Fisher Scientific Pvt. Ltd, Product No.-28035, Mumbai, Maharashtra, India),) were added. The mixture was incubated for 1 hour at RT. The final volume was adjusted to 5 ml using distilled water and the absorbance was measured at 510 nm using a UV–Visible spectrophotometer (Model DU 730, Beckman Coulter Inc., Brea, California, USA). A calibration curve was prepared using ascorbic acid as the standard. Each assay was conducted in triplicate (n = 3) to ensure reproducibility. Scavenging activity IC₅₀ calculated using;

where, A₀ = control without sample, A₁ = sample, A₂ sample without sodium salicylate.

Nitic Oxide scavenging activity.

Nitic oxide activity was assessed according to [21] with minor modifications. A 10 mM sodium nitroprusside solution in phosphate-buffer saline (PBS) (molecular biology grade, SRL-Sisco Research Laboratories Pvt. Ltd., P Code-78529, SDS version 2.0, Taloja, Maharashtra, India) was used as the reaction mixture. 2 ml of the reaction mixture was mixed with 200 µl fucoidan extract of different concentrations. The mixture was incubated at 37°C for 1hr. Then, 500 µl of Griess reagent (extra pure 98%, SRL-Sisco Research Laboratories Pvt. Ltd., P Code-28042, SDS version 4.0, Taloja, Maharashtra, India) was added. The percent inhibition was calculated by taking absorbance at 540 nm and IC₅₀ values were determined as described before. Measurements were taken in triplicate (n = 3) and results were expressed as mean ± standard deviation. PBS was used as the blank and BHA (Butylated hydroxyanisole) (purity 98% GC grade, SRL-Sisco Research Laboratories Pvt. Ltd., P Code-60004, SDS version 4.0, Taloja, Maharashtra, India) was taken as positive control.

FRAP assay.

The assay was carried out according to [22]. Fresh FRAP reagent was prepared by mixing 300 mM sodium acetate anhydrous (purity 98% SQ grade, Thermo Fisher Scientific India Pvt. Ltd. P Code- 20125, Mumbai, Maharashtra, India) buffer (pH 3.6), TPTZ (2,4,6-tri (2-pyridyl)-s-triazine) solution (extra pure 99% AR grade, SRL-Sisco Research Laboratories Pvt. Ltd., P Code-25793, SDS version 4.0, Taloja, Maharashtra, India) and 20 mM FeCl₃ anhydrous (pure 98%, SRL-Sisco Research Laboratories Pvt. Ltd., P Code-72287, Taloja, Maharashtra, India) in a 10:1:1 ratio. The mixture was incubated in dark for 10 minutes. Then, 60 µl of fucoidan extract, 90 µl of sodium acetate buffer and 1.5 ml of FRAP reagent were mixed, followed by incubation in dark for 30 minutes. The analysis was performed in triplicates (n = 3) to ensure consistency of results. A calibration curve was prepared using ferrous sulfate (FeSO₄.7H₂O) as the standard. Absorbance was measured at 593 nm, using acetate buffer as blank. The antioxidant capacity was determined using the regression equation y = 0.464x − 0.0114 (R² = 0.9996) where x is the absorbance at 593 nm and y is the concentration varied from 0.1 - 2mM and Results were expressed as mM Fe(II)/g of extract.

Inhibition assay for α-D-glucosidase enzyme activity.

Assay was determined in independent triplicates (n = 3) as per the modified method of [23,24]. Different fucoidan concentrations (320 µl aliquots each) were mixed with 60 µl of α-D-glucosidase enzyme solution (ex. Yeast, SRL-Sisco Research Laboratories Pvt. Ltd., P Code- 75551, SDS version 1.0, Taloja, Maharashtra, India) and 500 µl of Sodium Phosphate Buffer (SPB). Incubation was carried out at 37°C for 15 min in a water bath to allow enzyme-fucoidan interaction. Then, 320 µl of 2 mM PNP-G (p-Nitrophenyl-β-D-Galactopyranoside) (extra pure 98%, SRL-Sisco Research Laboratories Pvt. Ltd., P Code-12735, Taloja, Maharashtra, India) was added after incubation, and the mixture was incubated again for 10 min at 37°C. Subsequently, 500µl of sodium carbonate (NaCO₃) (purity-99.5% SQ grade, Thermo Fisher Scientific Pvt. Ltd, Product No.-27555, SDS version-2.0, Mumbai, Maharashtra, India) was added to stop the reaction. Absorbance was measured at 405 nm using a UV–Visible spectrophotometer (Model DU 730, Beckman Coulter Inc., Brea, California, USA). A blank was prepared for each fucoidan concentration by replacing both enzyme and substrate with SPB. A control was prepared by replacing enzyme solution with SPB. Acarbose was used as the reference standard to compare the inhibitory activity.

Inhibition assay for α-amylase activity.

The α-amylase inhibitory activity was determined according to the modified method [18,23,24]. Briefly, 200 µL of fucoidan extract was mixed with 200 µL of α-amylase enzyme solution (ex. Porcine pancreas; SRL–Sisco Research Laboratories Pvt. Ltd., Product Code 28588, SDS version 1.0; Taloja, Maharashtra, India). The reaction mixture was incubated at 37 °C for 15 min. After incubation, 600 µL of starch solution was added to each tube as a substrate and the mixtures were further incubated for 5 min. The reaction was then terminated by adding 1 mL of 3,5-dinitrosalicylic acid (DNSA) reagent (purity 97%, synthesis grade; Loba Chemie Pvt. Ltd., Product No. 03480, SDS version 1.0; Mumbai, Maharashtra, India). The tubes were incubated in a boiling water bath at 100 °C to allow colour development followed by cooling to room temperature. The absorbance was measured at 540 nm using a UV–visible spectrophotometer (Model DU 730, Beckman Coulter Inc., Brea, California, USA). All analyses were conducted in triplicate. Appropriate blank and control samples were prepared under identical conditions to ensure accuracy. Acarbose served as the standard reference.

Cell viability assay (MTT detection assay).

The in vitro cytotoxicity of fucoidan was determined against MCF-7 cancer cells by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, purity 99.5% TLC grade, Sigma-Aldrich, Merck KGaA, USA, P Code: M2003) according to [25]. Cells were seeded in triplicates in 96-well plates and cultured. Ten microliters (10 µl) of fucoidan (2 mg/ml) were added to each well and incubated for 48 h. Afterwards, 10 µl of MTT reagent (Invitrogen) was added per 100 µl of medium to each well, and incubated for 4 h at 37°C. The resulting formazan crystals were then dissolved in 50 µl of dimethyl sulfoxide (DMSO) (99%, EMPLURA®, Merck Life Sciences Pvt. Ltd, P code: 1.16743.0521, Mumbai, Maharashtra, India) and incubated at 37°C for 15 min. The optical density was measured using a microplate reader (Bio-Rad Laboratories, Inc., Model 680, USA) at 540 nm. All analyses were conducted in triplicate (n = 3). Doxorubicin was used as the standard.

Results

FT-IR analysis

The FT-IR spectra of fucoidan were depicted in Fig 1. Table 1 showing observed peaks of FT-IR analysis of Fucoidan and comparison with standard Fucoidan [30–32] The band at approximately 1024 cm^-1 corresponds to the C-O vibrations of the polysaccharides. The absorption around 1249 cm^-1 indicates the O = S = O stretching of sulfates, serving as a marker for sulfated polysaccharides. The absorption at 1595 cm^-1 corresponds to carbonyl (C = O) groups of uronic acid, while the stretching at 1404 cm^-1 corresponds to C-O within the –COOH group. The band around 3270 cm^-1 corresponds to O-H stretching, a common peak for sugars. The C-H stretching band at approximately 2900 cm^-1 corresponds to the methyl groups of fucose. The band at approximately 815 cm^-1 corresponds to C-O-S bending vibrations of sulfate groups, primarily located at the C-2, C-3 or C-4 position.

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Table 1. Table showing observed peaks of FT-IR analysis of Fucoidan and comparison with Standard Fucoidan (from literature).

https://doi.org/10.1371/journal.pone.0346712.t001

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Fig 1. FT-IR Spectra of crude Fucoidan from Sargassum wightii.

https://doi.org/10.1371/journal.pone.0346712.g001

NMR analysis

The ¹H NMR spectrum of fucoidan shown in Fig 2. The signal at 4.54 ppm corresponds to 3-linked α-L-fucose, a major fucoidan component while the signal at 1.6 ppm represents the methyl protons of fucopyranose residues [33]. The signal at 1.2 ppm indicates the presence of alkane protons in methyl groups. The signal at 2.4 ppm corresponds to protons attached to alkyl groups linked to sulfonyl moieties whereas the signals between 3–4 ppm corresponds to methoxy-linked proton. The signal at 3.5 ppm is attributed to α-L rhamnosyl residues.

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Fig 2. ¹H NMR analysis of crude fucoidan from Sargassum wightii.

https://doi.org/10.1371/journal.pone.0346712.g002

Determination of Fucose content in Fucoidan

Fucose content (% dry weight) was analyzed among six different species of brown seaweeds and was found to vary from 36.85–89.68%. The lowest fucose sugar content (36.85% ± 0.02) was observed in Sargassum tenerrimum, followed by 41.71% ± 0.01 in Sargassum cinereum while the highest content was recorded in Turbinaria conoides with 89.68% ± 0.01 (Fig 3).

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Fig 3. Fucose, Sulphate and Uronic acid content of fucoidan.

Values are presented as Mean ± SD (n = 3). Different superscript letters (a, b, c, d, f and e) indicate significant differences at p < 0.05 by one-way ANOVA followed by Duncan’s post hoc comparison test.

https://doi.org/10.1371/journal.pone.0346712.g003

Determination of antioxidant capacity

DPPH scavenging activity.

DPPH scavenging activity of six fucoidan samples from brown seaweeds is shown in Fig 4. It was observed that Sargassum cinereum and Sargassum wightii exhibited the highest DPPH activity with IC₅₀ of 0.342 ± 0.02 mg/ml and 0.347 ± 0.012 mg/ml respectively, while Sargassum tenerrimum 1.47 mg/ml ± 0.035 showed the lowest activity.

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Fig 4. Antioxidant activity (IC₅₀) of crude fucoidan through DPPH, ABTS, HRSA, NO.

Values are presented as Mean ± SD (n = 3). Different superscript letters (a, b and c) indicate significant differences at p < 0.05 by one-way ANOVA followed by Duncan’s post hoc comparison test.

https://doi.org/10.1371/journal.pone.0346712.g004

ABTS scavenging activity.

ABTS scavenging activity of six fucoidan samples is depicted in Fig 4. Sargassum wightii (IC₅₀ 1.09 ± 0.034 mg/ml) exhibited the highest activity followed by Sargassum cinereum (IC₅₀ 1.18 ± 0.027 mg/ml), while Padina boergesenii (IC₅₀ 1.82 ± 0.021 mg/ml) showed the lowest radical eliminating capacity.

HRSA scavenging activity.

Fig 4 shows the scavenging activities of six fucoidan samples against the hydroxyl radicals. The highest activity was observed in Sargassum wightii; (IC₅₀ of 1.23 ± 0.012 mg/ml) and in Sargassum cinereum (IC₅₀ 1.26 ± 0.01 mg/ml). Padina boergesenii (IC₅₀ 1.69 ± 0.021 mg/ml) and Sargassum tenerrimum (IC₅₀ 1.80 ± 0.021 mg/ml) showed the lowest activity.

Nitric Oxide scavenging activity.

Nitric Oxide assay of fucoidan samples is shown in Fig 4. Turbinaria conoides (IC₅₀ 1.12 ± 0.018 mg/ml) exhibited the highest activity, followed by Sargassum cinereum (IC₅₀ 1.20 ± 0.022 mg/ml) and Sargassum wightii (IC₅₀ 1.29 ± 0.013 mg/ml), while Padina tetrastromatica (IC₅₀ 1.33 ± 0.013 mg/ml) showed the least activity.

FRAP assay.

FRAP activity of all fucoidan samples is depicted in Fig 5. Sargassum cinereum (33.17 ± 0.02mM Fe (II)/g extract) showed the highest FRAP activity, followed by Sargassum tenerrimum (18.21 ± 0.02 Fe (II)/g extract), Padina tetrastromatica (17.36 ± 0.025 Fe (II)/g extract), Padina boergesenii (17.37 ± 0.024 Fe (II)/g extract) and Turbinaria conoides (17.18 ± 0.027 Fe (II)/g extract), while the lowest activity was observed in Sargassum wightii (13.84 ± 0.19 mM Fe (II)/g extract).

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Fig 5. FRAP activity of crude fucoidan.

Values are presented as Mean ± SD (n = 3). Different superscript letters (a, b, c and d) indicate significant differences at p < 0.05 by one-way ANOVA followed by Duncan’s post hoc comparison test.

https://doi.org/10.1371/journal.pone.0346712.g005

Enzymatic activity

α- amylase inhibitory activity.

The inhibitory potential of fucoidan against α- amylase and α-D-glucosidase using PNP-G as the substrate was screened. Inhibitory activity of fucoidan from all six seaweeds against α- amylase is shown in Fig 6. The highest activity was found in Sargassum wightii having IC₅₀ 0.042 ± 0.02 mg/ml making it the most effective, followed by Sargassum tenerrimum (IC₅₀ = 0.059 ± 0.01 mg/ml) and, Sargassum cinereum (IC₅₀ = 0.0728 ± 0.02 mg/ml). Padina tetrastromatica has (IC₅₀ = 1.152 ± 0.01 mg/ml) and was the least effective.

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Fig 6. Enzymatic activity of crude fucoidan against α- amylase and α- glucosidase.

Values are presented as Mean ± SD (n = 3). Different superscript letters (a, b, c, d, e and f) indicate significant differences at p < 0.05 by one-way ANOVA followed by Duncan’s post hoc comparison test.

https://doi.org/10.1371/journal.pone.0346712.g006

α-D-glucosidase inhibitory activity.

Inhibitory activity of fucoidan from all six seaweeds against α-D-glucosidase is shown in Fig 6. Sargassum cinereum was found to be the most effective (IC₅₀ = 0.22 ± 0.013 mg/ml), followed by Turbinaria conoides (IC₅₀ = 0.229 ± 0.022 mg/ml), Sargassum wightii (IC₅₀ 0.45 ± 0.02 mg/ml) while Padina tetrastromatica (IC₅₀ = 1.32 ± 0.19 mg/ml) showed the least effectiveness.

Cytotoxicity of fucoidan on MCF7 cancerous cells

The anticancer activity of fucoidan (at 2 mg/mL) on MCF-7 breast cancer cell lines is depicted in Fig 7. In this study, among the species; cells treated with fucoidan extracted from Padina boergesenii exhibited the highest toxicity, with only 11.43% ± 0.044 cell viability. This was followed by Turbinaria conoides (21.5% ± 0.143), Sargassum wightii (33.26% ± 0.103), Padina tetrastromatica (34.84% ± 0.174) and Sargassum tenerrimum (37.27% ± 0.119). The least toxicity was observed in Sargassum cinereum, which showed 42.96% ± 0.199 viability. Viable cells actively convert MTT into the colored compound formazan. The results were statistically significant (p < 0.05) when compared with other samples.

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Fig 7. Cytotoxic effect of crude fucoidan.

Values are presented as Mean ± SD (n = 3). Different superscript letters (a, b and c) indicate significant differences at p < 0.05 by one-way ANOVA followed by Duncan’s post hoc comparison test.

https://doi.org/10.1371/journal.pone.0346712.g007

Statistical analysis

Results were expressed as the mean of the replicates ± standard deviation. One-way single factor ANOVA was performed on the dataset to analyze various antioxidant; enzymatic and chemical activities compare the significant differences in the means of different treatments at p < 0.05. Results are shown in Table 2. It was found to be highly significant with p-value of 8.44E-11, 0.038711 and 3.41E-08 which were much lower than 0.05; a similar result was found for the F-value, where F > F_critical. The coefficient of variation (CV%) values indicated acceptable experimental precision across all parameters, with relatively low variability observed for fucose sugar, sulphate content, uronic acid, antioxidant, and enzyme inhibitory activities among the studied seaweed species. Overall, Turbinaria conoides and Padina spp. showed more consistent results (lower CV%), while comparatively higher variability was observed in α-amylase inhibition and cell viability across species (Table 3). The higher CV% values (>60%) observed in some species may be attributed to the inherent variability of the MTT assay, including its sensitivity to differences in metabolic activity and cell seeding density, as well as species-specific cellular responses.

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Table 2. (A) Single factor ANOVA analysis of dataset for antioxidant activities. (B) Single factor ANOVA analysis of dataset for enzymatic activities. (C) Single factor ANOVA analysis of dataset for chemical activities.

https://doi.org/10.1371/journal.pone.0346712.t002

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Table 3. Coefficient of variation (CV%) for monosaccharide fucose sugar, sulphate content, uronic acid, sulphate/fucose ratio, yield, antioxidant activities (DPPH, ABTS, HRSA, NO, and FRAP assays), enzyme inhibitory activities (α-amylase and α-glucosidase IC₅₀), and cell viability (%) of crude fucoidan extracted from different brown seaweed species.

https://doi.org/10.1371/journal.pone.0346712.t003

Discussion

Brown seaweeds represent a valuable source of bioactive compounds with wide-ranging pharmacological and nutraceutical potential. Among these, fucoidan a complex sulfated polysaccharide is emerging biological and therapeutic relevance due to its diverse biological activities. Fucoidan exhibits potent antioxidant, anti-inflammatory, antiviral, anticoagulant and anticancer properties with its antioxidant capacity being particularly important for neutralizing free radicals and mitigating oxidative stress. Beyond its antioxidant function, it also modulates immune responses, promotes wound healing and supports cardiovascular health, underscoring its value in pharmaceutical and cosmeceutical formulations. The bioactivity of fucoidan however, varies considerably among species and is largely influenced by factors such as algal source, structural complexity, degree of sulfation, acetylation, methylation, monosaccharide composition, uronic acid content and extraction technique. Therefore, optimizing extraction parameters is crucial to obtain fucoidan with consistent purity and biological efficacy. In the present study, EDTA-based extraction proved effective yielding fucoidan fractions devoid of detectable polyphenols or EDTA residues. This high degree of purity ensures the reliability of subsequent structural and biological evaluations, thereby establishing a strong foundation for assessing the therapeutic potential of fucoidan derived from Indian brown seaweeds.

FT-IR analysis revealed functional groups characteristic of fucoidan, aligning to a significant degree, with those of earlier conducted research works by [34] and [20]. Absorption bands between 2853–2989 cm ⁻ ¹ indicated CH stretching of the pyranose ring and the C6 group in fucose and galactose. S = O groups, identified in the spectra of P. pavonica (1195–1237 cm ⁻ ¹) and S. vulgare (1210–1280 cm ⁻ ¹), are characteristic of fucoidan [35]. The ¹H NMR spectrum displayed peaks at 3.92 ppm (attributable to β-d-galactose) and 3.5–4.5 ppm (ring protons indicating sulfate groups), consistent with the study [34].

The fucose content of fucoidan showed marked variation among the examined brown seaweeds, reflecting clear species-specific differences in polysaccharide composition. It was further influenced by extraction method, seasonal variation and collection site. Sargassum tenerrimum showed the lowest fucose proportion (36.85% ± 0.02), whereas Turbinaria conoides contained the highest level (89.68% ± 0.01). These findings are consistent with earlier studies reporting 39.04% in S. tenerrimum [36], 59.1% in S. illicifolium [37], 53% in Sargassum wightii as reported [38]. The fucose content in T. conoides was notably higher than that in T. decurrens (59.3%) [39], highlighting interspecific variation and further support that Turbinaria species serve as richer sources of fucose-based fucoidan. Since higher fucose content has been linked to greater antioxidant, anticoagulant activities, given fucose’s crucial role in the structural integrity and bio functionality of fucoidans. Conversely, the comparatively lower fucose content in Sargassum species may indicate a more heterogeneous polysaccharide composition, potentially influencing their overall bioactivity. However, when comparing species-specific bioactivities, it is important to consider that variations in molecular weight (MW) may also contribute to the observed differences, as MW is a key determinant of fucoidan functionality. Low sugar levels are preferred in functional foods for their low glycemic impact [16].

Sulfate content and its positional distribution are key determinants of fucoidan bioactivity [29]. They enhance the molecule’s negative charge density, facilitating interactions with enzymes such as α-amylase which can inhibit enzyme activity, suppress cell proliferation and contribute to antioxidant properties [30]. In the present study, sulfate levels varied notably among species, ranging from 6.31 ± 0.01% in Sargassum tenerrimum to 22.83 ± 0.016% in Turbinaria conoides. It corroborates previous finding including Undaria pinnatifida (22.83 ± 1%) [15], Laminaria japonica (27.95%) [12] Padina boergesenii (17.72 ± 10.25%) [28] and S. fluitans (7.56%) [33].

Uronic acid in fucoidan contributes significantly to its anticoagulant potential by interacting with plasma proteins and modulating blood lipid profiles [34]. In the present study, uronic acid levels were relatively low, with Padina boergesenii containing 1.91 ± 0.015% (w/w) and Sargassum tenerrimum 1.35 ± 0.016% (w/w). These values align with previous findings for Undaria pinnatifida (4.06 ± 0.44%) [15], Turbinaria ornata (7.8%) [31] and Dictyota dichotoma (2.25 ± 0.80%) [35]. The observed variation suggests that although it is present in smaller amounts compared to fucose or sulfate, uronic acids play a vital structural role, contributing carboxyl groups that enhance the electrostatic interactions of fucoidan with biomolecules involved in coagulation pathways.

The available DPPH‐radical scavenging data show significant differences among these seaweeds. Notably, Sargassum cinereum was by far the most potent DPPH scavenger: its (IC₅₀ = 0.342 ± 0.019 mg/ml) followed by S. wightii. This implies that S. cinereum’s polysaccharides are much more effective at donating hydrogen to neutralize DPPH. Such enhanced activity often correlates with a higher sulfate‐to‐fucose ratio in the fucoidan, since sulfate groups strongly boost radical‐scavenging potential. Previous studies show lower activity in S. binderi (IC₅₀ = 2.01 ± 0.29 mg/ml) [40], S. hystrix (IC₅₀ = 2200.23 ± 1014.77 ppm) [41] likewise, L. japonica has only moderate activity [42]. Other studies include 72.59 ± 0.32% inhibition for S. wightii [43] and 55.62% for P. boergesenii at 1 mg/ml [44]. The differences likely arise from their distinct polysaccharide and other content as well as possibly differing secondary metabolites.

ABTS scavenging activity among the tested species ranged between 1.09–1.82 mg/ml, with Sargassum wightii showing a strong IC₅₀ = 1.09 ± 0.034 mg/ml. The markedly higher activity of S. wightii suggests a richer pool of antioxidant constituents, possibly due to a higher phenolic or sulfate content contributing to enhanced radical-quenching ability. However, similar reports found in studies done in Padina tetrastromatica (IC₅₀ = 0.5 ± 0.04 mg/ml) and Turbinaria conoides (IC₅₀ = 0.1 ± 0.03 mg/ml) [40]. Environmental factors, seasonal changes and extraction conditions may also account for the observed variability in ABTS radical scavenging efficiency among brown seaweeds.

Hydroxyl radical scavenging plays a crucial role in protecting cellular components against oxidative stress-induced damage [41]. In the present study, the observed activity was comparable to that reported for Sargassum hystrix (IC₅₀ = 2360.07 ± 536.93 ppm) [32], but lower than that of S. ilicifolium, which exhibited an IC₅₀ of 209.99 ± 0.25 µg/ml [25]. Comparatively, Dictyota species showed about 8% inhibition at 0.5 mg/ml [45]. while Undaria pinnatifida displayed stronger inhibition (76.97 ± 1.69% at 2 mg/ml) [14]. This indicates interspecific variability in hydroxyl radical scavenging efficiency within species. Such differences are likely influenced by variations in structural polysaccharides, degree of sulfation, phenolic composition, environmental and extraction-related factors that modulate the availability of hydrogen-donating compounds.

Nitric oxide (NO) scavenging activity was most pronounced in P. boergesenii (IC₅₀ = 1.27 ± 0.023 mg/mL). These findings agree with earlier reports for the same species. (IC₅₀ = 5061.88 µg/mL) [28], S. polycystum (IC₅₀ = 5.36 ± 0.08 mg/mL) [39]. This enhanced activity may be attributed to variations in extraction method, bioactive composition or seasonal factors influencing metabolite yield.

Ferric reducing antioxidant power (FRAP), reflecting the electron-donating ability of the extracts, ranged from 17–33 mM Fe (II)/g. S. cinereum exhibited the highest reducing capacity (33.17 ± 0.02 mM Fe (II)/g), indicating strong reductive potential compared to P. tetrastromatica (IC₅₀ = 135 ± 0.1 mg/mL) and T. conoides (IC₅₀ = 241 ± 0.1 mg/mL) [40]. These results align with previous reports such as 71.38 ± 6.14 µM/g in S. hystrix [32], 24.81 ± 2.07 µg/mL in U. pinnatifida [23] and 4.82 ± 0.11 µM/g in P. australis [46], highlighting species-dependent variations in antioxidant potency. Saccharina wightii exhibited strong DPPH radical scavenging activity but relatively low FRAP values, reflecting the different mechanisms measured by these assays. DPPH primarily evaluates the ability of compounds to donate hydrogen atoms to neutralize free radicals, whereas FRAP measures the electron-donating capacity to reduce ferric ions. The structural features of S. wightii fucoidan such as accessible sulfate groups and hydrogen-donating moieties favor radical scavenging (DPPH) but may not efficiently participate in electron transfer reactions required for FRAP. This demonstrates that antioxidant activity of fucoidans is assay-dependent and influenced by specific structural characteristics rather than total sulfation alone.

Fucoidan extracted from S. wightii exhibited strong α-amylase inhibitory activity (IC₅₀ = 0.042 ± 0.02 mg/mL) as compared to the standard drug acarbose (IC₅₀ = 0.108 ± 0.017 mg/mL) as well as U. pinnatifida (IC₅₀ = 0.190 ± 0.005 mg/mL) [24] and A. nodosum (IC₅₀ = 0.12–4.64 mg/mL) [44]. Comparable inhibitory potential has been reported in studies in T. ornata (IC₅₀ = 33.6 ± 0.7 µg/mL) [21] and S. polycystum (IC₅₀ = 2.30 ± 0.05 mg/mL) [39]. The pronounced enzyme inhibition observed in S. wightii may be attributed to its high sulfate content and molecular configuration, which enhance binding affinity toward the enzyme’s active site.

Similarly, α-D-glucosidase inhibition was most potent in S. cinereum (IC₅₀ = 0.22 ± 0.013 mg/mL) comparing of acarbose (IC₅₀ = 1 ± 0.3 mg/mL) [45]. Comparable activities have been documented in E. maxima (IC₅₀ = 0.27–0.31 mg/mL) [46], S. wightii (IC₅₀ = 132 ± 0.02 µg/mL) [26], S. polycystum (IC₅₀ = 3.71 ± 0.04 mg/mL) [39] and Saccharina japonica (IC₅₀ = 153.27 ± 22.89 µg/mL) [47]. The present findings also align closely with U. pinnatifida (IC₅₀ = 0.137 ± 0.012 mg/mL) [43], suggesting an inhibitory mechanism among brown algal fucoidans. Fucoidan from S. wightii selectively inhibited α-amylase, likely due to accessible sulfate groups and flexible backbone structure, whereas S. cinereum fucoidan preferentially inhibited α-glucosidase, reflecting a more compact structure and distinct sulfate positioning. These findings highlight that enzyme-specific inhibition is governed by species-dependent fucoidan structural features. Overall, the potent α-amylase and α-glucosidase inhibition in S. wightii and S. cinereum highlights their strong antidiabetic potential through carbohydrate-hydrolyzing enzyme suppression.

The present study demonstrates the potent anticancer activity of fucoidan derived from Padina boergesenii and Turbinaria conoides. When compared with the standard anticancer drug doxorubicin (10 µg/mL), which reduced MCF-7 cell viability to 7–9% after 48 h, P. boergesenii fucoidan demonstrated comparable cytotoxic potential, while other extracts showed moderate inhibition. Padina boergesenii exhibited the highest cytotoxicity despite moderate sulfation, suggesting that cytotoxic activity is influenced by multiple structural features beyond total sulfate content. Factors such as molecular weight, monosaccharide composition, fucose content, branching, and specific sulfate positioning can enhance interactions with cancer cell membranes or intracellular targets, leading to stronger bioactivity. Therefore, moderate overall sulfation does not preclude potent cytotoxic effects if other structural attributes favor biological activity. This aligns with previous reports indicating that structural nuances, not only sulfate density, determine fucoidan’s anticancer potential. Notably, many studies have reported comparable cytotoxic effects. A study by [47] found that 48 h treatment with crude Undaria pinnatifida fucoidan (1 mg/mL) left only 2.6%viable MCF-7 cells. Fucoidan from S. polycystum inhibited MCF-7 breast cancer cell proliferation (IC₅₀ ≈ 50 ± 0.08 µg/mL) [48]. The cytotoxic effect of fucoidan from P.boergesenii on human cervical carcinoma cells demonstrated a significant percentage of inhibition 54% at 40 μg/mL for [28]. Fucoidan-Based Gold Nanoparticles derived from T. decurrens and S. cinereum exhibited significant activity against HepG2 cells, with IC50 values of 449.5 µg mL − 1 and 337.6 µg mL − 1, respectively [49].

The efficacy of fucoidan is largely governed by its structural characteristics, particularly sulfate content, molecular weight and fucose-to-sulfate ratio which modulate its cellular uptake and interaction with cancer cell receptors [50]. Studies have shown that fucoidan induces both extrinsic and intrinsic apoptotic pathways through activation of caspases-8 9, 3, cytochrome c release and downregulation of anti-apoptotic [51]. In particular, fucoidan has been shown to induce reactive oxygen species (ROS)-mediated apoptosis in human bladder cancer cells by inhibiting telomerase activity and suppressing the PI3K/Akt signaling cascade, leading to programmed cell death [52].

Overall, our data demonstrate that fucoidan’s cytotoxicity toward MCF-7 cells is both species-specific and statistically significant, with extracts from P. boergesenii and T. conoides showing the most promising activity, approaching the effect of standard doxorubicin. This enhanced activity may arise from apoptotic and oxidative mechanisms associated with higher sulfate substitution. Such ROS-dependent cytotoxicity emphasizes the dual role of fucoidan as both a redox regulator and a signal modulator in cancer suppression.

In addition to the in vitro findings, our molecular docking analysis further supports these observations by demonstrating favorable binding affinities of fucoidan toward key apoptosis- and survival-related targets. The predicted interactions with proteins involved in the PI3K/Akt pathway and caspase activation provide a mechanistic rationale for the experimentally observed cytotoxic effects. However, while docking offers valuable insights into potential ligand-protein interactions, these computational predictions require further validation through molecular dynamics simulations, target-specific enzymatic assays, and gene/protein expression studies.

Therefore, future work should focus on integrating advanced in silico approaches, including molecular dynamics and binding free energy calculations, with in vitro and in vivo validation to comprehensively elucidate the structure–activity relationship of fucoidan. Such studies will strengthen the mechanistic understanding of its anticancer potential and support its development as a multi-targeted natural therapeutic agent.

PCA analysis

Principal Component Analysis (PCA) is an advanced multivariate statistical reduction technique that transforms complex datasets into a more interpretable form by reducing multiple variables into a few principal components (PCs) that explain the maximum variance within the data. In this study, PCA and biplot analyses were performed to understand the interrelationship between the antioxidant and enzymatic parameters of six brown seaweed species (Fig 8A). The biplot revealed that FRAP activity, fucose, and sulfate content were positively correlated with all Sargassum species, whereas the remaining parameters showed negative associations. Among the species, Padina boergesenii exhibited the highest fucose content, Turbinaria conoides had the maximum sulfate content, and Sargassum cinereum showed the strongest FRAP activity. In contrast, ABTS activity displayed the least variance across species.

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Fig 8. (A) PCA Biplot analysis representing the variation in different bioactivities in fucoidan.

(B) PCA Score plot analysis representing the variation in different bioactivities in fucoidan. (C) Dendrogram showing Agglomerative Hierarchical Clustering. (D) Boxplot Analysis.

https://doi.org/10.1371/journal.pone.0346712.g008

The score plot (Fig 8B) further demonstrated that FRAP and fucose contributed most significantly to data variance, while other variables were moderately correlated. Notably, DPPH IC₅₀ values exhibited a negative correlation with both FRAP activity and fucose content, suggesting that higher antioxidant potential corresponds to lower IC₅₀ values. Similarly, α-amylase inhibitory activity showed a positive correlation with FRAP and fucose, indicating a possible linkage between antioxidant and enzyme inhibitory mechanisms. The clustering of FRAP, fucose, and sulfate along the same principal component suggests that sulfate-rich fucoidan fractions may directly contribute to enhanced antioxidant performance, as the sulfation degree is known to increase electron-donating and radical-scavenging capacity in brown algal polysaccharides. Principal component analysis (PCA) was conducted using bioactivity data exclusively, encompassing the measured biological activity parameters of the fucoidan samples. Due to the functional relationships among these bioactivity variables, they exhibited strong inter-correlations across samples. As a result, the first principal component (PC1) captured the majority of the variance, explaining 96.29% of the total variance. Such a high contribution of PC1 is common in PCA models with a limited set of highly correlated functional variables, where a single dominant factor largely governs the overall biological response. The remaining variance was distributed among the subsequent components (PC2 and higher), which contributed only marginally to the differentiation of samples. It should be noted that this high dominance of PC1 limits the discriminatory power of the PCA. Incorporating structural variables (e.g., molecular weight, branching patterns) in future analyses would likely produce a more informative and discriminating model.

Agglomerative Hierarchical Clustering (AHC) was employed to classify the seaweed species based on their overall biochemical similarity (Fig 8C). The dendrogram revealed close clustering among T. conoides, P. boergesenii, and S. wightii, all of which were characterized by relatively high sulfate and fucose levels, correlating with elevated FRAP and α-amylase inhibitory activities. This grouping highlights a shared biochemical profile driven by sulfate-rich fucoidan composition, which is strongly linked to antioxidant potency. In contrast, S. cinereum appeared as a distinct cluster, reflecting its higher FRAP activity but differing sulfation profile, suggesting that its antioxidant capacity may derive from non-sulfated or polyphenolic components. Meanwhile, P. tetrastromatica and S. tenerrimum formed a secondary cluster with lower sulfate and fucose contents, aligning with their comparatively moderate antioxidant responses.

Additionally, the box-and-whisker plot (Fig 8D) provided a visual summary of data distribution and variability. The highest degree of variability was observed in T. conoides, followed by P. boergesenii, S. wightii, and S. cinereum, while S. tenerrimum and P. tetrastromatica exhibited the least data dispersion, indicating relatively uniform biochemical profiles among their replicates. Overall, the PCA and AHC results together demonstrate that the sulfate and fucose composition of fucoidans serves as a biochemical determinant influencing both antioxidant and enzyme inhibitory activities across brown seaweed species, confirming the close biochemical–functional correlation among Sargassum and related genera.

Conclusion

This study highlights the structural diversity and potent bioactivity of fucoidan extracted from various brown seaweeds along the Indian coast. Characterization through FT-IR and ¹H NMR spectroscopy confirmed the presence of functional groups associated with bioactive fucoidan, including sulfate esters and monosaccharides such as fucose and galactose. The composition particularly fucose and sulfate content varied significantly across species, influencing their antioxidant, antidiabetic, and anticancer properties.

Among the studied species, Sargassum wightii, S. cinereum, and Padina boergesenii demonstrated superior biological activities. Fucoidan from S. wightii exhibited strong α-amylase and ABTS radical scavenging activity, while S. cinereum showed notable DPPH, FRAP, and α-D-glucosidase inhibitory activities. These activities are likely attributed to the favorable sulfate-to-fucose ratios and molecular configurations of the polysaccharides. Additionally, the low sugar and uronic acid content in some species further supports the potential of fucoidan as a functional food component with low glycemic impact.

The findings confirm the therapeutic potential of brown seaweed-derived fucoidan, supporting its application in nutraceuticals, functional foods, and pharmaceuticals. Further research on structure-function relationships and seasonal or geographical variability will strengthen its commercial viability and facilitate targeted bioactive formulation.

Supporting information