Novel tyrosinase inhibitory peptides from Kaempferia parviflora protein hydrolysate with melanin suppression and mechanistic insights from B16F10 cells and zebrafish models

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Black ginger ( Kaempferia parviflora ) is a widely used Thai medicinal rhizome, but its protein-derived fraction has not yet been widely studied for potential anti-melanogenic activity. In this work, we used bromelain to obtain the protein hydrolysate from black ginger and conducted activity-guided fractionation to obtain tyrosinase-inhibitory peptides (TIPs) with mechanistic and cellular validation. Ultrafiltration generated five molecular-weight fractions, among which the <0.65 kDa fraction exhibited the greatest mushroom tyrosinase inhibition in both monophenolase and diphenolase assays. Activity was further enriched via RP-HPLC, and NanoLC–ESI–MS/MS analysis followed by Mascot database-assisted identification and manual inspection of the fragmentation spectra identified two short peptides, YGRGPIQ (YQ-7) and YNYGPAGQA (YA-9). Synthetic peptides confirmed intrinsic activity, with YA-9 showing stronger inhibition than YQ-7 (IC 50 = 0.56 ± 0.02 mM vs 1.20 ± 0.03 mM). Enzyme kinetics indicated competitive inhibition for both peptides in mono- and diphenolase systems, while molecular docking supported active-site-associated binding through hydrogen bonding and hydrophobic contacts near the catalytic pocket. In B16F10 cells, MTT metabolic activity remained above 80% across the tested concentration range, while both peptides reduced cellular tyrosinase activity and melanin content. RT-qPCR and Western blot analyses further showed concentration-dependent modulation of melanogenesis-related markers, with the strongest and most consistent suppression observed for TYR and TRP-1 and more variable responses for MITF and TRP-2. YA-9 was selected for zebrafish embryo testing based on the more consistent molecular profile in B16F10 cells. Immersion exposure showed low developmental toxicity, but no statistically significant depigmentation was detected. Overall, these results identify black ginger as a new source of sequence-defined TIPs and suggest that YA-9 and YQ-7 are promising leads, but their limited in vivo depigmentation indicates that delivery optimization or structural modification is needed to translate the cellular effects.