Impact of sequential extraction on structure and antioxidant activity of fucoidan from Turbinaria spp

Sequential extraction has recently emerged as an alternative strategy for the comprehensive utilization of brown seaweed biomass, enabling the recovery of multiple bioactive compounds, including fucoidan. However, its influence on fucoidan structure and bioactivity remains insufficiently understood. In this study, fucoidan was isolated from Turbinaria spp. using a sequential extraction approach involving a prior removal of protein, and the impact of this process on fucoidan structure and bioactivity was systematically evaluated and compared to fucoidan obtained from a conventional acidic extraction. The conventional fucoidan crude extract (CFCE) yielded higher fucoidan content (5.28%) and contained greater levels of sulfate, uronic acid, and protein material than the sequential fucoidan crude extract (SFCE). In contrast, SFCE exhibited higher carbohydrate content (62.9%) and markedly reduced protein levels (3.4%), indicating greater carbohydrate enrichment and reduced protein co-extraction. Although both extraction strategies yielded fucoidan with a fuco-galactose backbone dominated by α-(1 → 3)- and α-(1 → 4)-L-fucose and β-(1 → 3)-D-galactose linkages, with comparable sulfation patterns after purification, only minor differences in glycosidic linkage composition were observed between extraction methods. Functionally, CFCE, SFCE, and their respective fractions exhibited no cytotoxicity towards Caco-2 cells, while the purified fractions significantly attenuated hydrogen peroxide-induced intracellular reactive oxygen species. Metabolomic analyses revealed distinct intracellular metabolic responses associated with fucoidan treatment, including metabolite changes associated with glutathione metabolism and phenylalanine and tyrosine metabolism. Overall, sequential extraction enabled the recovery of purified and structurally defined fucoidan with significant bioactivity. These findings support sequential extraction as a promising strategy for integrated seaweed biorefinery applications and efficient biomass utilization.