Tyrosinase inhibition and enzyme kinetics of red rice bran protein peptides: Mechanism, identification, in silico exploration, and experimental validation

Red rice (Oryza sativa L.) was recorded to have anti-melanogenic activity, but its active compounds were unknown. This study was intended to evaluate the tyrosinase inhibitory capacity of rice bran protein (RRBP) hydrolysates and obtain a novel tyrosinase-inhibitory peptide. RRBP was extracted with 0.2% alkali solution and hydrolyzed with alcalase (RRBP-A), pancreatin (RRBP-P), neutral (RRBP-N) and papain (RRBP-M), respectively. The results demonstrated that RRBP-A hydrolysate showed highest DPPH and ABTS radical scavenging abilities, FRAP value, tyrosinase-inhibitory effect and copper chelating activity. A total of 449 peptides were identified from RRBP-A by LC-MS/MS analysis. After a multi-step virtual screening process, Trp-Phe-Gly (WFG), Trp-Trp-Ala (WWA), Leu-Cys-Trp (LCW), Phe-Leu-Pro (FLP) and Tyr-Phe-Pro (YFP) were screened from RRBP-A. Compared with other peptides, LCW had higher DPPH (IC₅₀, 109.5 ± 6.36 μg/mL), ABTS radical scavenging ability (IC₅₀, 28 ± 1.41 μg/mL), FRAP value (349.07 ± 6.51 μmol/L), tyrosinase-inhibitory activity (IC_50, 153.5 ± 4.95 μg/mL), and copper chelating activity (IC_50, 1245.3 ± 83.1 μg/mL). HPLC analysis further confirmed that LCW acted as tyrosinase inhibitor. Molecular docking studies showed that LCW could bind to tyrosinase protein via both Cu400 and Cu401 sites, and other tyrosinase active sites. Additionally, LCW had an effective inhibitory effect on melanin production in mouse melanoma cell line (B16-F10 cells) by suppressing tyrosinase activity. The enzyme kinetic results exhibited that LCW had a mixed-type inhibition of tyrosinase. These results demonstrated that LCW had great potential on tyrosinase inhibitory effect and melanin production, indicating that LCW might be served in skin health care.