Given the increasing demand for skin whitening and health products, tyrosinase inhibitors have garnered significant attention in the food, pharmaceutical, and cosmetic industries. However, conventional inhibitors like arbutin, hydroquinone, and kojic acid often face limitations due to issues with solubility, stability, and safety concerns. This has spurred a major research focus on discovering novel, safe, and natural tyrosinase inhibitors.
Bioactive peptides have emerged as promising candidates due to their excellent bioactivities, high absorption rates, and favorable safety profiles, demonstrating potential as enzyme inhibitors, including tyrosinase. Locusts, particularly Locusta migratoria, represent a rich and sustainable source of high-quality protein and essential nutrients, holding promising potential for various industrial applications. Compared to other animals, locusts offer advantages such as high feed conversion efficiency, rapid growth, strong environmental adaptability, minimal space requirements, low water consumption, and reduced greenhouse gas emissions, aligning with sustainable development principles. Locusta migratoria stands out among edible insects for its higher protein content, better digestibility, and more balanced essential amino acid profile, making it a particularly suitable candidate for developing bioactive peptides. Despite these benefits, there have been no prior reports on the production of tyrosinase inhibitory peptides derived from locust protein hydrolysates. This study aimed to bridge this gap by investigating defatted locust powder as a source of bioactive peptides with tyrosinase inhibitory activity.
Methods
The study utilized enzymatic hydrolysis of defatted Locusta migratoria protein to generate peptides, which were subsequently separated into fractions based on molecular weight using ultrafiltration. The specific peptides were then identified through LC-MS/MS, and their molecular mechanisms of tyrosinase inhibition were elucidated using molecular docking analysis. Additionally, the safety and bioactivity of the peptides were evaluated at the cellular level using human normal HEK-293T cells and B16 melanoma cells.
Key Findings
• Enzymatic hydrolysis significantly enhanced tyrosinase inhibition, with a non-hydrolyzed protein showing only a 6.5 ± 0.85% inhibition rate, compared to up to 38.22% for certain enzyme hydrolysates.
• Optimal enzymatic hydrolysis conditions were determined to be a material-to-liquid ratio of 1:15, an enzyme concentration of 3000 U/g, a hydrolysis time of 2 hours, and a hydrolysis temperature of 58 °C using Longda acidic protease.
• Peptides with molecular weights below 1 kDa exhibited the strongest tyrosinase inhibitory effect, showing a 53.00 ± 0.65% inhibition rate at 10 mg/mL, outperforming other fractions.
• Structural analysis revealed that peptides with more exposed aromatic amino acids (phenylalanine, tyrosine, tryptophan), weaker hydrogen bonding, and lower α-helix content exhibited enhanced inhibitory activity.
• LC-MS/MS identified 1,108 peptide sequences, predominantly from Vitellogenin B (34.78%) and Vitellogenin A (34.53%), with over 80% of peptides having molecular weights below 1500 Da.
• The <1 kDa fraction contained a high proportion of glycine, alanine, leucine, and isoleucine, with approximately 62.62% of isolated peptides containing amino acids capable of inhibiting tyrosinase.
• Kinetic studies confirmed that the peptides act as mixed-type, reversible inhibitors, suggesting they bind to both the free enzyme and the enzyme-substrate complex, thus decreasing enzyme activity.
• Molecular docking identified key interactions for tyrosinase inhibition, primarily through hydrogen bonds and hydrophobic interactions with critical residues in the enzyme’s active site, preventing substrate binding. Key binding residues included His61, His85, His259, His263, and His296.
• The peptide DF9 exhibited the most interactions with the binding pocket (eighteen hydrophobic and eight hydrogen bonds) and the lowest binding energy of -7.9 kcal/mol.
• The peptides showed low cytotoxicity in human normal HEK-293T cells, maintaining over 75% cell viability at concentrations up to 8 mg/mL, demonstrating superior biocompatibility compared to kojic acid.
• In B16 melanoma cells, the peptides exhibited a significant 37.26% inhibition rate of tyrosinase activity at 2 mg/mL, comparable to kojic acid at a lower concentration. While copper ion chelation activity was observed for the peptides at 6 mg/mL (over 80% chelation rate), molecular docking indicated no direct interaction of the identified peptides with copper ions at the active site.
This study presents novel and compelling insights into the development of tyrosinase inhibitory peptides derived from Locusta migratoria protein hydrolysates, a previously unexplored source for this application. The research highlights the remarkable efficacy of smaller peptides (below 1 kDa), demonstrating their superior inhibitory activity linked to specific structural features like exposed aromatic amino acids, weaker hydrogen bonding, and lower α-helix content. The detailed mechanistic studies, including kinetic analysis confirming mixed-type, reversible inhibition, and molecular docking identifying critical binding residues and interaction types (hydrogen bonds and hydrophobic interactions), provide a robust theoretical basis for their inhibitory action.
Crucially, the peptides exhibited a high safety profile with low cytotoxicity in HEK-293T cells and demonstrated significant tyrosinase inhibition in B16 melanoma cells, indicating their biological relevance and potential for practical applications. This research positions locust-derived peptides as a promising natural alternative to conventional tyrosinase inhibitors, addressing long-standing concerns regarding the solubility, stability, and safety of existing options.
For topical science, these findings are particularly significant. The potent tyrosinase inhibitory activity, coupled with their low cytotoxicity and natural origin, makes these locust peptides highly attractive for incorporation into cosmetic formulations aimed at skin whitening and treating hyperpigmentation disorders such as freckles, melasma, and age spots. Furthermore, their demonstrated ability to influence melanin production in B16 melanoma cells suggests potential applications in anti-melanoma therapies, offering new avenues for dermatological research and product development. Beyond cosmetics and therapeutics, their inhibitory properties also open doors for their use as natural preservatives in food systems, further expanding their impact. Future research could focus on in vivo studies to validate their efficacy and safety in human skin models and explore specific peptide sequences for targeted applications.
Link to the study: https://tinyurl.com/msp9j8nm
