Ultrasonic assisted enzymatic extraction, structural characterization, and tyrosinase inhibition mechanism of phlorotannins from Sargassum horneri

Abstract

Phlorotannins, recognized for their efficacy and safety as natural tyrosinase inhibitors, have posed challenges due to their limited extraction efficiency and the elusive nature of their mechanism of action. Here, we developed an ultrasonic-assisted enzymatic extraction (UAEE) protocol that significantly enhanced phlorotannin recovery from Sargassum horneri, achieving a total phenolic content (TPC) of 21.51 mg PGE/g DW. Subsequent purification using solvent extraction and Sephadex LH-20 yielded high-purity alginate phlorotannins (SHP), exhibiting potent DPPH radical scavenging and tyrosinase inhibitory activities, with IC₅₀ values of 22.71 and 26.90 μg/mL, respectively. Structural elucidation based on NMR and MS data suggested that SHP predominantly comprises fucophloroethols, with a degree of polymerization ranging from 3 to 8. Reciprocal plot analysis indicated that SHP inhibits tyrosinase activity through a competitive inhibition mechanism, while fluorescence spectroscopy revealed a dynamic quenching effect of SHP on tyrosinase. Molecular docking studies further elucidated that van der Waals forces and hydrophobic interactions are the primary forces governing the interaction between SHP and tyrosinase. Cellular experiments demonstrated the significant inhibitory effects of SHP on both melanin production and tyrosinase activity in B16-F10 cells. This research presents promising lead compounds for the development of novel tyrosinase inhibitors, while also providing theoretical and methodological foundations for the comprehensive utilization of Sargassum horneri.