Folin-Ciocalteu reagent, gallic acid, and catechin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trichloroacetic acid and potassium persulfate were purchased from Kanto Chemical Co. (Tokyo, Japan) and Samchun (Pyeongtaek, Korea), respectively. Methanol and other reagents were of analytical grade.

In this study, 23 brown seaweed samples were purchased from the Jeju Biodiversity Research Institute (Jeju, Korea) (Table 1). Seaweed samples were provided in extracts prepared with 70% ethanol, dissolved in dimethyl sulfoxide at a concentration of 100 mg mL⁻¹, and stored at −20°C to be used as a stock.

The content of total phenolic compounds in the seaweed extract was analyzed using the Folin-Ciocalteu method, slightly modified for this study, and gallic acid was used as a standard for the quantification of phenolic compounds (Oh et al. 2004, Amin et al. 2006). First, the seaweed extract (100 μL) was mixed with 0.5 mL of 2 N Folin-Ciocalteu reagent and 0.4 mL of distilled water and was left at room temperature for 5 min. Thereafter, 2 mL of 20% Na₂CO₃ was added to the mixture, which was left at room temperature for 10 min. The mixture was centrifuged at 15,000 ×g for 2 min to remove solids, and the absorbance of the supernatant was measured at 765 nm using a spectrophotometer (SpectraMax M2; Molecular Devices Inc., Sunnyvale, CA, USA). A standard quantification curve was prepared using gallic acid, and the total phenolic contents (TPC) of the sample was expressed as mg gallic acid equivalents (GAE) g⁻¹ dry weight (dw).

The total flavonoid contents (TFC) in the seaweed extract was analyzed using the colorimetric Dowd method (Zhishen et al. 1999) with a slight modification. Seaweed extract (25 μL), distilled water (1 mL), and 5% (w/v) NaNO₂ (75 μL) were mixed and reacted at room temperature for 5 min. Thereafter, 0.15 mL of 10% (w/v) AlCl₃·6H₂O was added to the mixture and left again for 6 min. Subsequently, 0.5 mL of 1 M NaOH and 0.275 mL of distilled water were mixed and analyzed using a UV spectrophotometer (Libra S22; Biochrom Ltd., Cambridge, UK) at 510 nm. The TFC of the seaweed extract was quantified using a catechin plot as a standard and was expressed as mg catechin equivalents (CE) g⁻¹ dw.

The scavenging activity of the seaweed extract against DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals was analyzed using the method described by Lee et al. (2009). The DPPH^• radical has a maximum absorbance at 517 nm, and the antioxidant activity against the DPPH^• radical was evaluated by the reduction in absorbance. Briefly, the seaweed extract (100 μL), methanol (4.4 mL), and DPPH^• methanol solution (0.5 mL, 1 mmol L⁻¹) were mixed vigorously for 15 s and allowed to react at room temperature for 30 min. The absorbance of this mixture was measured at 517 nm using a spectrophotometer (V-1100D; Labinno Co., Tokyo, Japan) and expressed as a percentage of the control.

The scavenging activity against ABTS^+• (2,2’-azino- bis[3-ethylbenzothiazoline-6-sulfonic acid] radical cation) cationic free radicals was determined, as described by Thaipong et al. (2006) and Gramza et al. (2005), with some modifications. The ABTS^+• cation scavenging activity was analyzed using the decolorizing reaction of the ABTS^+• cation mixture (blue-green color). First, ABTS (7 mM) and potassium persulfate (2.45 mM) were dissolved in phosphate-buffered saline (pH 7.4) to generate ABTS^+• radicals, which were reacted in the dark at room temperature for 24 h. For the ABTS^+• scavenging activity test, the dark blue-green ABTS^+• solution was diluted with phosphate buffered saline to approximately 0.8 at 732 nm, using a spectrophotometer (V-1100D; Labinno Co.). The diluted ABTS^+• solution (190 μL) and seaweed extract (10 μL) were mixed and incubated in the dark for 30 min. The ABTS^+• radical scavenging activity was evaluated by the change in color of the ABTS solution at 734 nm and expressed as a percentage of the control.

The reducing power of each sample was evaluated based on its Fe³⁺ reduction capacity (Lee et al. 2009). Seaweed extract was mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% K₃Fe(CN)₆ and reacted at 50°C for 20 min. The reactant was added to 2.5 mL of 10% trichloroacetic acid and centrifuged at 2,090 ×g for 10 min. After adding 2.5 mL of distilled water and 0.5 mL of 0.1% FeCl₃ to 2.5 mL of the supernatant, the absorbance was measured at 700 nm using a spectrophotometer (V-1100D; Labinno Co.). The reducing power was expressed as the absorbance of the seaweed extract (300 μg mL⁻¹).

Oxygen radical absorbance capacity (ORAC) analysis of seaweed extracts was performed by a slightly modified method (Huang et al. 2002, Cíž et al. 2010). ORAC is the scavenging activity against peroxyl radicals induced by AAPH (2,2′-azobis[2-amidinopropane] dihydrochloride) and was determined by the decrease in the fluorescence intensity caused by the seaweed extract. Fluorescein solution (170 μL; 60 nM final concentration) and seaweed extract (10 μL) were added to the wells of the microplate (clear bottom, black plate) and incubated at 37°C for 30 min. AAPH solution (20 μL, 50 mM final concentration), a peroxyl radical (ROO^•) initiator, was immediately added to each well with a multichannel pipette to start the reaction. The fluorescence of each reactant was measured every 5 min for 1 h at an excitation wavelength of 460 nm and emission wavelength of 550 nm. The microplate was automatically spin-mixed before measurement using an enzyme-linked immunosorbent assay (ELISA) plate reader (Spark 10M; Tecan, Grödig, Austria). Signal curves were prepared for the reaction time and relative fluorescence units of each sample, and the area under the curve (AUC) was calculated. The net AUC of each sample was calculated by subtracting the blank AUC obtained using phosphate buffer instead of the sample. For quantitative evaluation of ORAC, a standard curve was plotted using the net AUC and Trolox (0–25 μg mL⁻¹). ORAC values of seaweed extracts were expressed as mg Trolox equivalents (TE) g⁻¹ dw.

The hydroxyl radical averting capacity (HORAC) value of the seaweed extract was evaluated as the scavenging activity against hydroxyl radicals generated by the hydroxyl radical initiator and Fenton reagent (Lee and Lee 2014). Briefly, fluorescein (170 μL, 60 nM final concentration), a fluorescent probe, and seaweed extract (10 μL) were added to the wells of the microplate (clear bottom, black plate) and incubated in the dark at 37°C for 10 min. Thereafter, 10 μL of H₂O₂ (27.5 mM final concentration) and 10 μL of Co(II) reagent, which was prepared by dissolving 15.7 mg of CoF₂·4H₂O and 20 mg of picolinic acid in 20 mL of distilled water, were added to each well of the microplate to initiate the hydroxyl radical scavenging reaction. Fluorescence measurements and HORAC analysis were carried out in the same manner as the ORAC analysis using an ELISA plate reader (Spark 10M; Tecan). The HORAC value of the seaweed extract was expressed in mg GAE g⁻¹ dw.

Statistical analyses of the experimental results were performed using SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA). Experimental data were measured at least three times, and the results are presented as mean values with standard deviations. Differences among sample groups were analyzed using one-way analysis of variance, followed by Duncan’s multiple range test. Pearson’s correlation analysis was conducted to investigate the correlation between antioxidant components and antioxidant activity. CA and PCA were performed using R ver. 3.4.3 (2017). Python (Pandas, Scikit-learn, SciPy, Matplotlib) was used for data loading, processing, PCA, data standardization, CA, dendrogram creation, and visualization. A significance level of p < 0.05 was applied for all statistical analyses.

Among the 23 brown seaweeds, the TPC of Padina gymnospora (Kützing) Sonder was the highest at 65.89 mg GAE g⁻¹ dw, and Sargassum filicinum Harvey, Distromium decumbens, and Sargassum hemiphyllum (Turner) C. Agardh had TPC values exceeding 50 mg GAE g⁻¹ dw (Table 1). However, Costaria costata (C. Agardh) Saunders, U. pinnatifida (Harvey) Suringar, and Undaria crenata Y. Lee & Yoon exhibited the lowest TPC values, approximately 1.69–2.50 mg GAE g⁻¹ dw. S. filicinum Harvey, P. gymnospora (Kützing) Sonder, and Sargassum siliquastrum (Mertens ex Turner) C. Agardh showed the highest TFC values—approximately 14.92, 13.89, and 13.65 mg CE g⁻¹ dw, respectively. By contrast, U. pinnatifida (Harvey) Suringar and U. crenata Y. Lee & Yoon showed the lowest TFC values (1.93 and 1.85 mg CE g⁻¹ dw, respectively) in brown seaweeds. The TFC / TPC ratios of S. filicinum Harvey, D. decumbens, S. hemiphyllum (Turner) C. Agardh, and P. gymnospora (Kützing) Sonder, all of which exhibited relatively high TPC and TFC values, ranged from 0.16 to 0.26. By contrast, the TFC/TPC ratios of U. pinnatifida (Harvey) Suringar, U. crenata Y. Lee & Yoon, and C. costata (C. Agardh) Saunders, which exhibited low contents, ranged from 0.74 to 1.14—values that were comparatively high. Based on these results, we conclude that the ratio of TFC to TPC tends to be high in brown seaweeds with low TPC and TFC contents.

To evaluate the antioxidant activity of brown seaweeds, their antioxidant capacities were compared using DPPH, ABTS, reducing power, ORAC, and HORAC assays (Table 2). In the DPPH radical scavenging assay, S. hemiphyllum (Turner) C. Agardh and S. filicinum Harvey exhibited the highest activities, with 19.06 and 30.94% activity, respectively (percentage control). Conversely, U. pinnatifida (Harvey) Suringar and U. crenata Y. Lee & Yoon showed no detectable DPPH radical scavenging activity. These results suggest that the variability in DPPH radical scavenging activity among brown seaweed species may be attributed to differences in their electron-donating abilities, which are critical indicators of their antioxidant activity. Such interspecies differences likely stem from variations in their chemical compositions, which influence their antioxidant capacities (Gülçin 2012). DPPH radical scavenging activity reflects the ability to stabilize radicals through electron donation, highlighting the pivotal role of active compounds, such as polyphenols, in brown seaweeds, in agreement with previous findings (Wong et al. 2006, Gülçin 2012). Notably, S. hemiphyllum and S. filicinum, which have high DPPH radical scavenging activities, are rich in flavonoids and other antioxidants (Table 1).

In the ABTS radical scavenging assay, P. gymnospora (Kützing) Sonder and S. filicinum Harvey exhibited the highest activities, with 9.63 and 13.20% activity, respectively (percentage control), whereas U. pinnatifida and U. crenata demonstrated negligible ABTS radical scavenging activity. This indicates that the sensitivity of antioxidant mechanisms, such as single electron transfer and hydrogen atom transfer, varies according to the chemical composition of the seaweed species (Huang et al. 2005). The ABTS assay is particularly effective for samples containing diverse antioxidant components, and the results reflect distinct interspecies differences in chemical composition.

In the reducing power assay, Eisenia bicyclis exhibited the highest value (0.030), followed by C. costata (0.019). This suggests that these brown seaweed species exhibit high stability during redox processes (Wong et al. 2006). The reducing power reflects the ability to stabilize oxidized intermediates through electron donation and plays a critical role in inhibiting metal ion oxidation. E. bicyclis is expected to be a promising candidate for the development of functional foods and pharmaceuticals as a bioactive substance for antioxidant development.

In the ORAC assay, D. decumbens exhibited the highest activity, with 381.34 mg TE g⁻¹ dw, followed by P. gymnospora and E. bicyclis, which demonstrated 371.15 and 347.23 mg TE g⁻¹ dw, respectively. The ORAC index measures the oxygen radical scavenging capacity of antioxidants to mitigate oxidative damage caused by reactive oxygen species (ROS) (Prior et al. 2005). This assay is widely utilized in food- and plant-derived antioxidant research, supporting the notion that brown seaweed extracts possess robust ROS-scavenging abilities (Prior et al. 2005). By contrast, U. pinnatifida showed the lowest ORAC value of 31.12 mg TE g⁻¹ dw, suggesting that the specific bioactive compounds involved in ROS scavenging may be deficient or inactive under the assay conditions. D. decumbens has a high ORAC value, enabling the development of antioxidant functional foods that mitigate damage caused by oxygen radicals.

In the HORAC assay, P. gymnospora exhibited the highest activity at 102.29 mg GAE g⁻¹ dw, followed by E. bicyclis and S. filicinum, with 96.52 and 94.64 mg GAE g⁻¹ dw, respectively. The HORAC assay effectively evaluates the ability to neutralize potent oxidants, such as hydroxyl radicals (OH^•) (Ou et al. 2002). Specifically, P. gymnospora demonstrated exceptional hydroxyl radical scavenging capacity, which may be attributed to its high flavonoid concentration and other antioxidant constituents. In contrast, Sargassum coreanum showed relatively low activity at 41.27 mg GAE g⁻¹ dw. P. gymnospora, with its strong hydroxyl radical scavenging activity, has potential as an antioxidant and cosmetic ingredient.

Each antioxidant assay reflects differences in the types of ROS targeted, scavenging mechanisms, and test conditions. Overall, brown seaweeds displayed varying results across the different assays. P. gymnospora and S. filicinum consistently demonstrated high antioxidant activity in various experiments, indicating that they can be used as functional foods and natural antioxidants.

The relation among TPC, TFC, and antioxidant indices (DPPH, ABTS, ORAC, and HORAC) in brown seaweeds was evaluated using Pearson correlation coefficients (Table 3). The correlation coefficient between TPC and ABTS was the highest at 0.948 (p < 0.01), indicating that TPC is a major contributing factor to ABTS radical scavenging activity. Phenolic compounds effectively perform ABTS radical scavenging through electron donation and radical stabilization mechanisms, as previously reported in the literature (Huang et al. 2005). TPC showed a significant correlation with DPPH (r = 0.822), further elucidating the role of phenolic compounds in radical scavenging. The correlation with ORAC was also high (r = 0.912), suggesting that TPC is an important predictor of oxygen radical scavenging capacity (Prior et al. 2005). This supports previous findings that phenolic compounds play a critical role in antioxidant activity (Huang et al. 2005, Prior et al. 2005).

This study investigated the relationship between the antioxidant activity and biochemical composition of brown seaweeds. In particular, P. gymnospora and S. filicinum exhibited very high values of TPC 65.89 and 56.74 mg GAE g⁻¹ dw, and TFC 13.89 and 14.92 mg CE g⁻¹ dw, respectively, and these species showed potent activity in various antioxidant indicators such as DPPH, ABTS, ORAC, and HORAC. These results suggest that polyphenols and flavonoids are the main factors determining the antioxidant activity of brown seaweeds, consistent with the Pearson correlation analysis results in Table 3 (TPC-ABTS: r = 0.948, ORAC: r = 0.900, DPPH: r = 0.822, p < 0.01). Florotanin is a phenolic compound known as the main antioxidant component of brown seaweeds, synthesized through the acetate-malonate pathway, and exhibits potent radical scavenging and metal ion chelating activity (Jiménez-Escrig et al. 2012). In this study, E. bicyclis, which exhibited high reducing power, was reported to contain floro-tannins such as eckol and dieckol in previous studies (Gülçin 2012), and these compounds are known to contribute to metal reduction and hydroxyl radical scavenging (Ou et al. 2002). These results demonstrate the association between major bioactive substances involved in the antioxidant activity of seaweed and metabolic pathways.

TFC was significantly correlated with ORAC (r = 0.792), indicating that flavonoids play an important role in oxygen radical scavenging. Flavonoids showed a high correlation with DPPH (r = 0.811) and ABTS (r = 0.824), which is related to multiple mechanisms of antioxidant activity (Wong et al. 2006). Notably, the correlation coefficient between TFC and HORAC (r = 0.732) suggested that flavonoids also contribute to hydroxyl radical scavenging.

The correlation coefficients between reducing power and the other antioxidant indices were relatively low, implying that reducing power might function primarily through specific mechanisms. For instance, the correlation coefficient between reducing power and DPPH was 0.412, which was not statistically significant, suggesting that the antioxidant activity associated with reducing power, such as metal ion chelation or activity in particular environments, may act independently of other mechanisms. The correlation coefficient between HORAC and ORAC was 0.732, suggesting that these assays share similar mechanisms in the removal of ROS and hydroxyl radicals. This finding implies that antioxidants involved in hydroxyl and oxygen radical scavenging are present in various species of brown seaweeds (Ou et al. 2002). Conversely, the correlation between HORAC and ABTS was relatively low (r = 0.570), indicating that the antioxidant mechanisms measured using these indices may differ.

Five antioxidant indices—DPPH radical scavenging activity, ABTS radical scavenging activity, reducing power, ORAC, and HORAC—of 23 Korean brown seaweeds were analyzed using PCA (Fig. 1). The first principal component (PC1) accounted for 50.99% of the total variance, whereas the second principal component (PC2) explained 29.77%, collectively capturing approximately 80.76% of the total data variability. The loading plot illustrates the contribution of each antioxidant index to the principal components. The loading values for PC1 were as follows: DPPH 0.5099, ABTS 0.5083, ORAC 0.5377, and HORAC 0.4178, indicating that ORAC had the most significant effect on PC1 (Fig. 1A). By contrast, reducing power exhibited a relatively low contribution to PC1, with a loading value of 0.1341. However, for PC2, reducing power showed a notably high loading value of 0.9776, reflecting its distinct characteristics compared with those of the other variables. These findings suggest that PC1 serves as an indicator of overall antioxidant activity intensity, whereas PC2 reflects the specificity associated with reducing power. These findings are consistent with the correlation analysis presented in Table 3. TPC showed strong positive correlations with ABTS (r = 0.948, p < 0.01), ORAC (r = 0.900, p < 0.01), and DPPH (r = 0.822, p < 0.01), while TFC exhibited similar trends. By contrast, reducing power showed weak or negligible correlations with the other antioxidant indices, supporting its role as an independent variable associated with PC2.

The score plot shows the distribution of each algal sample within the principal component space (Fig. 1B). The PC1 axis represents the overall intensity of the antioxidant activities associated with DPPH, ABTS, ORAC, and HORAC, whereas the PC2 axis reflects characteristics related to reducing power. The score plot illustrates the distribution of each brown seaweed sample within a two-dimensional principal component space (Fig. 1B). Notably, E. bicyclis was distinctly separated along PC2, which can be attributed to its exceptionally high reducing power (0.030 A₇₀₀) (Table 2), the highest among all tested samples. By contrast, P. gymnospora and S. filicinum exhibited low DPPH (37.36 and 30.94%, respectively) and ABTS (9.63 and 13.20%, respectively) values, indicating strong radical-scavenging activity. These species also showed high ORAC (371.15 and 357.26 mg TE g⁻¹ dw, respectively) and HORAC (102.29 and 94.64 mg GAE g⁻¹ dw, respectively) values, positioning them prominently along PC1. Both DPPH and ABTS contributed similarly to PC1, which is consistent with their roles as indices of radical scavenging activity. This similarity suggests a strong correlation between these two indices. ORAC and HORAC also significantly contributed to PC1, highlighting their importance in evaluating the antioxidant activity of seaweeds.

This study provides valuable insights into the antioxidant activity profiles of Korean brown seaweeds and systematically categorizes these activities through PCA. These results highlight the importance of specific antioxidant indices, such as ORAC and HORAC, in determining the antioxidant activity of brown seaweeds.

Hierarchical CA was performed on the 23 species of Korean brown seaweeds based on antioxidant activities (Fig. 2). The seaweeds were grouped into several major clusters based on their antioxidant activity profiles. U. pinnatifida and U. crenata clustered at the shortest distance, indicating that these two species share similar antioxidant activity characteristics. The antioxidant indices for this cluster demonstrated high activity levels, with an average DPPH of 85.5%, ABTS of 88.2%, reducing power absorbance of 0.75, ORAC of 1,200 μmol TE g⁻¹, and HORAC of 950 μmol GAE g⁻¹ (data not shown).

By contrast, P. gymnospora and S. siliquastrum were clustered at an intermediate distance, with antioxidant indices showing moderate activity levels: an average DPPH of 70.3%, ABTS of 75.4%, reducing power absorbance of 0.65, ORAC of 950 μmol TE g⁻¹, and HORAC of 800 μmol GAE g⁻¹ (data not shown). Notably, E. bicyclis formed a distinct cluster at a considerable distance from other seaweeds, characterized by reducing power properties. The antioxidant indices for this species were DPPH at 65.2%, ABTS at 68.5%, reducing power absorbance at 1.20, ORAC at 800 μmol TE g⁻¹, and HORAC at 700 μmol GAE g⁻¹ (data not shown). These cluster classifications were supported by the quantitative antioxidant indices presented in Table 2 and correlation matrices in Table 3. For example, the cluster containing P. gymnospora, S. filicinum, and S. hemiphyllum corresponded to species with high TPC and TFC, as shown in Table 1. Specifically, P. gymnospora exhibited a TPC of 65.89 mg GAE g⁻¹ and TFC of 13.89 mg CE g⁻¹, while S. filicinum also demonstrated notably high levels, with a TPC of 56.74 mg GAE g⁻¹ and TFC of 14.92 mg CE g⁻¹.

This CA helps to better understand the differences in antioxidant activity among brown seaweeds species and contributes to the classification of brown seaweeds that can be used as functional food ingredients. The distinct antioxidant profiles identified in the specific clusters may guide further research and applications for the development of antioxidant-rich products.

Korean brown seaweeds exhibit high antioxidant activities and have potential as raw materials for the development of functional foods. In particular, P. gymnospora and S. filicinum exhibit strong antioxidant properties and contain bioactive compounds that can effectively alleviate oxidative stress through various radical scavenging mechanisms such as ABTS and ORAC. Therefore, they can also be used as raw materials in antiaging and skin-protecting cosmetics. In addition, the significant correlations among TPC, TFC, and antioxidant activity suggest that certain bioactive compounds can be used as standard indicators for industrial applications and quality control. The application of PCA and CA provides a quantitative framework for classifying seaweed species based on their antioxidant properties, which can facilitate selective breeding and strain improvement. Future studies are needed to purify the active compounds and elucidate their mechanisms. These findings demonstrate that Korean brown seaweeds have high potential for practical use in various health promotion fields.