Pearl and nacre powders have been valuable traditional Chinese medicines with whitening properties for thousands of years. We utilized a high-temperature and high-pressure method along with compound enzyme digestion to prepare the enzymatic hydrolysates of nacre powder of Pinctada martensii (NP-PMH). The peptides were identified using LC–MS/MS and screened through molecular docking and molecular dynamics simulations. The interactions between peptides and tyrosinase were elucidated through enzyme kinetics, circular dichroism spectropolarimetry, and isothermal titration calorimetry. Additionally, their inhibitory effects on B16F10 cells were explored. The results showed that a tyrosinase-inhibitory peptide (Ala-His-Tyr-Tyr-Asp, AHYYD) was identified, which inhibited tyrosinase with an IC₅₀ value of 2.012 ± 0.088 mM. The results of the in vitro interactions showed that AHYYD exhibited a mixed-type inhibition of tyrosinase and also led to a more compact enzyme structure. The binding reactions of AHYYD with tyrosinase were spontaneous, leading to the formation of a new set of binding sites on the tyrosinase. The B16F10 cell-whitening assay revealed that AHYYD could reduce the melanin content of the cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that AHYYD could be widely used as a tyrosinase inhibitor in whitening foods and pharmaceuticals.

For thousands of years, pearl and nacre powders have been important traditional Chinese medicines known for their skin whitening effects. To prepare the enzymatic hydrolysates of Hyriopsis cumingii nacre powder (NP-HCH), complex enzymatic hydrolysis by pineapple protease and of neutral protease was carried out after the powder was pre-treated with a high-temperature and high-pressure method. The peptides were identified using LC-MS/MS and picked out through molecular docking and molecular dynamics simulations. Subsequently, the tyrosinase inhibitory and antioxidant properties of novel tyrosinase inhibitory peptides were investigated in vitro. In addition, the enzymatic activity of tyrosinase in B16F10 cells as well as melanin content and antioxidant enzyme levels were also examined. The results showed that a tyosinase inhibitory peptide (Tyr-Pro-Asn-Pro-Tyr, YPNPY) with an efficient IC₅₀ value of 0.545 ± 0.028 mM was identified. The in vitro interaction results showed that YPNPY is a reversible competitive inhibitor of tyrosinase, suggesting that it binds to the free enzyme. The B16F10 cell whitening test revealed that YPNPY can reduce the melanin content of B16F10 cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that YPNPY could be widely used as a tyrosinase inhibitor in whitening foods and drugs.

Excessive melanin accumulation induced by UV exposure and hormonal changes poses significant challenges to skin health, necessitating safe and effective tyrosinase inhibitors. Pearl and nacre powder have long been utilized in traditional Chinese medicine for skin whitening. Our previous study identified a tyrosinase inhibitory peptide (Ala-His-Tyr-Tyr-Asp) from Pinctada martensii nacre in silico; however, chemical modification strategies for enhancing bioactive peptide efficacy remain underexplored. The present study investigated the structure-activity relationship of NP-TIP-1 (Tyrosinase Inhibitory Peptide of Nacre Powder-1) through three chemical modifications (C-terminal amidation, N-terminal acetylation, and N-terminal palmitoylation). C-terminal amidation significantly improved tyrosinase inhibitory potency, reducing the IC50 value from 2.012 ± 0.088 mM to 0.979 ± 0.028 mM, while retaining thermal stability and copper ion chelating capacity comparable to the native peptide. In B16F10 melanoma cells, NP-TIP-1-NH₂ (C-terminally amidated NP-TIP-1) exhibited no cytotoxicity at concentrations up to 600 μM and concentration-dependently suppressed intracellular tyrosinase activity and melanin content. Mechanistic investigation revealed that NP-TIP-1-NH₂ treatment downregulates the mRNA and protein expression of TYR and TRP-1 without affecting MITF (Microphthalmia-associated Transcription Factor) or TRP-2, suggesting a potential regulatory mechanism independent of MITF-mediated transcriptional control. Non-targeted metabolomics further identified nucleotide metabolism, purine metabolism, and glycine/serine/threonine metabolism as the most significantly perturbed pathways, with cytosine and GMP downregulated while guanosine and guanine were upregulated. This study establishes NP-TIP-1-NH₂ as a promising candidate for multifunctional food preservatives and nutraceuticals, providing a strategic framework for C-terminal amidation as a structure-activity optimization approach for marine-derived anti-melanogenic peptides.